BRCA1 intronic Alu elements drive gene rearrangements and PARP inhibitor resistance

Resistència terapèutica contra el càncer; Càncer d'ovarisResistencia terapéutica al cáncer; Cáncer de ovariosCancer therapeutic resistance; Ovarian cancerBRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.This work was supported by the US National Institutes of Health (NIH) Grants P50 CA083638 and 5P30 CA006927, as well as R01CA214799, Susan Komen CCRCR17499048, and OC130212 Department of Defense to N.J., and a Pilot Award and Nested Teal Postdoctoral Scholar supported Y.W. (OC140040). We thank Drs. Judith Balmaña, Cristina Saura and Joaquin Arribas for providing materials. PDXs were established thanks to the GHD-Pink program, the FERO Foundation and the Catalan Agency AGAUR [2017 SGR 540]. V.S. is supported by the Instituto de Salud Carlos III (CP14/00228) and ALG by a PERIS fellowship from the Departament de Salut, Generalitat de Catalunya (SLT002/16/00477). Clovis Oncology supplied rucaparib. We are grateful to FCCC Genomics, Cell Culture and Cell Sorting facilities. We thank Hsin Yao Tang at the Wistar Proteomics facility for help with mass spectrometry

Genetic Separation of BRCA1 Functions Reveal Mutation-Dependent Polθ Vulnerabilities

Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/Polq)-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq−/− cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin.

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR

BRCA1 Mutation-Specific Responses to 53BP1 Loss-Induced Homologous Recombination and PARP Inhibitor Resistance

Summary: BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brca1ΔC/ΔC mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1ΔC/ΔC;53bp1−/− mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance. : Using a Brca1ΔC mouse model and a panel of BRCA1 mutant cancer cell lines, Nacson et al. show that 53BP1 loss of function induced homologous recombination and PARP inhibitor resistance is suboptimal in the absence of hypomorphic BRCA1 proteins that retain the coiled-coil domain and ability to interact with PALB2. Keywords: BRCA1, 53BP1, homologous recombination, PARP inhibitors, resistanc

Genetic separation of Brca1 functions reveal mutation-dependent Polθ vulnerabilities

Abstract Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/Polq)-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq −/− cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies

The BRCA1-Δ11q Alternative Splice Isoform Bypasses Germline Mutations and Promotes Therapeutic Resistance to PARP Inhibition and Cisplatin

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR

The BRCA1-Δ11q alternative splice isoform bypasses germline mutations and promotes therapeutic resistance to PARP inhibition and cisplatin

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo. Furthermore, spliceosome inhibitors reduced BRCA1- Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance