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, Pol

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

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Conceived and designed the experiments: JJK OD RGH. Performed the experiments: JJK OD. Analyzed the data: JJK OD RGH. Wrote the paper: JJK OD RGH.Background and PurposeThe targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.MethodsA fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.ResultsThe purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.ConclusionThese results suggest that this treatment could be useful as a targeted therapy for breast cancer.Yeshttp://www.plosone.org/static/editorial#pee

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

Dissociation constant (<i>K_d</i>) of PNP-AV binding to cells.

<p>Dissociation constant (<i>K_d</i>) of PNP-AV binding to cells.</p

PNP-AV mechanism of action.

<p>Fludarabine is converted in serum to a dephosphorylated form by a 5′nucleotidase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-VandenNeste1" target="_blank">[37]</a>. PNP attached to the cell surface via annexin V and phosphatidylserine binding then cleaves the ribose-1-phosphate group, resulting in 2-fluoroadenine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-Lee1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-Mao1" target="_blank">[12]</a>. The freely diffusible molecule enters the cell and inhibits protein, RNA, and DNA synthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-Parker1" target="_blank">[13]</a>. Nucelotide-specific membrane transport carriers transport the dephosphorylated form across the cell membrane, where it is then phosphorylated into a cytotoxic triphosphate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-Pierige1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076403#pone.0076403-VandenNeste1" target="_blank">[37]</a>.</p

<i>In vitro</i> cytotoxicity of enzyme prodrug treatment.

<p>The effects of fludarabine, 2-fluoroadenine, and fludarabine converted to 2-fluoroadenine by PNP are shown for (a) non-confluent HAAE-1 cells, (b) MCF-7 cells, and (c) MDA-MB-231 cells. Groups that received PNP-AV were treated on days 0 and 3 of the study. Fludarabine and 2-fluoroadenine were administered daily. Viability was determined by the Alamar Blue assay on days 2, 4, and 6 (black, gray, and white bars, respectively), and each sample was represented as a percentage of untreated control on each day. Statistical analysis was performed with a one-way ANOVA test with data presented as mean ± SE (<i>n = </i>3). Statistical significance vs. untreated control on the same day is denoted by *(<i>p</i><0.001).</p

MCF-7 dissociation constant binding data.

<p>Specific binding (▴) was determined by subtracting total binding (•) in calcium supplemented medium from nonspecific binding (▪) in calcium deficient medium. Binding was quantified with biotinylated protein and HRP-conjugated streptavidin, developed with OPD. Data are presented as mean ± SE (<i>n = </i>3).</p

PNP-AV binding stability.

<p>Bound fusion protein over a 3-7 (♦), MDA-MB-231 (▴), and HAAE-1 (▪) cell lines. Data are presented as mean ± SE (<i>n = </i>3).</p