Homologous Recombination Activity In Cells Research Articles

Evaluating homologous recombination activity in tissues to predict the risk of hereditary breast and ovarian cancer and olaparib sensitivity.

Homologous recombination (HR) repairs DNA damage including DNA double-stranded breaks and alterations in HR-related genes results in HR deficiency. Germline alteration of HR-related genes, such as BRCA1 and BRCA2, causes hereditary breast and ovarian cancer (HBOC). Cancer cells with HR deficiency are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors and DNA-damaging agents. Thus, accurately evaluating HR activity is useful for diagnosing HBOC and predicting the therapeutic effects of anti-cancer agents. Previously, we developed an assay for site-specific HR activity (ASHRA) that can quantitatively evaluate HR activity and detect moderate HR deficiency. HR activity in cells measured by ASHRA correlates with sensitivity to the PARP inhibitor, olaparib. In this study, we applied ASHRA to lymphoblastoid cells and xenograft tumor tissues, which simulate peripheral blood lymphocytes and tumor tissues, respectively, as clinically available samples. We showed that ASHRA could be used to detect HR deficiency in lymphoblastoid cells derived from a BRCA1 pathogenic variant carrier. Furthermore, ASHRA could quantitatively measure the HR activity in xenograft tumor tissues with HR activity that was gradually suppressed by inducible BRCA1 knockdown. The HR activity of xenograft tumor tissues quantitatively correlated with the effect of olaparib. Our data suggest that ASHRA could be a useful assay for diagnosing HBOC and predicting the efficacy of PARP inhibitors.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Abstract Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

Interleukin-6 Adversely Impacts Genomic Stability Via Targeting Multiple Pathways in Multiple Myeloma

In our previous investigation, using whole exome sequencing, we have shown that multiple myeloma (MM) patients display a complex dynamic of clonal evolution and the number of mutations in patient samples correlate with overall and relapse free survival. These results highlight the importance of understanding the mechanisms driving genomic instability in MM. Investigating mechanisms underlying genomic instability, we have shown that dysregulated homologous recombination (HR), nuclease (especially apurinic/apyrimidinic related) and APOBEC deaminase activities contribute to genomic instability in MM. We have also demonstrated that bone marrow microenvironment (BMM) also contributes to genomic instability in MM. So here we have further investigated the soluble factors in BMM that may impact genomic integrity. We treated RPMI8226 MM cells with IL-6, IL-17 and TGFb and evaluated their impact on DNA breaks by monitoring the levels of gH2AX (a DNA break marker). Treatment with all 3 cytokines (IL-6, IL-17 and TGFb) caused DNA breaks, as demonstrated by increase in gH2AX, in MM as well as a solid tumor (esophageal cancer) cell lines. Importantly, among these cytokines, the exposure to IL-6 was associated with the highest induction of gH2AX expression. These observations were confirmed in additional MM cell lines (MM1S, H929, OPM2 and U266) treated with IL-6.Since we have previously demonstrated that elevated HR is a key mechanism of genomic instability in MM, we investigated the role of IL-6 in dysregulation of HR pathway by evaluating its impact on p-RPA32 (a marker of DNA end resection which is a decisive step in the initiation of HR), recombinase (RAD51) expression and HR activity (as assessed by a functional assay). Treatment of MM cells with IL-6 led to increased expression of p-RPA32 and RAD51 (as detected by Western blotting) as well as increased HR activity in MM cells. Increased HR activity was also observed following exposure of MM cells to IL-17, TGFb and IFNb, with the highest induction caused by IL-6. However, the combination of cytokines (IL-6, IL-17 and TGFb) led to a further (> 2-fold) increase in HR activity in these cells, indicating that these soluble factors may interact in inducing mechanisms underlying genomic instability in MM.Based on our data showing important role of apurinic/apyrimidinic (AP) nuclease in regulation of HR and genome stability in MM, we also evaluated the impact of IL-6 on abasic sites, the substrate of AP nuclease activity. An increase in the number of abasic sites (ranging from 1.7- to 2.3-fold increase) was observed with increasing concentration of IL-6 in MM1S cells. To further investigated the impact of IL-6 on number of micronuclei, used as marker of genomic instability. The treatment of MM cell lines with IL-6 for 48 hrs led to a dose-dependent increase (ranging from 2- to > 2.5-fold increase) in the number of micronuclei in MM1S and RPMI cells, indicating increased genomic instability. We have previously shown that inhibition of HR by RAD51 knockdown or ABL kinase inhibitor, nilotinib, significantly reduces genomic instability, as assessed by micronuclei assay as well as direct evaluation of impact on genomewide acquisition of copy number changes over time using SNP arrays. To investigate if HR inhibition can reverse IL-6-induced genomic instability, we treated MM cells with IL-6, nilotinib and combination of both and observed that inhibition of HR by nilotinib can reverse IL-6-induced increase in number of micronuclei (by 48±17%) in MM cells. Similarly, addition of anti-IL-6 antibody also reversed the IL6-induced DNA breaks (as assessed from gH2AX expression) in MM1S cells. Moreover, combination of nilotinib and anti-IL6 antibody resulted in maximum inhibition of IL6-induced DNA breaks in MM1S cells.Taken together, these data suggest that IL-6 significantly contributes to dysregulation of HR and genome stability in MM, and agents targeting HR and/or other mechanisms of genomic instability including anti-IL-6 antibody, have potential to reduce/delay genomic evolution and disease progression. DisclosuresMunshi:OncoPep: Other: Board of director.

Abstract PR02: Small molecules that specifically inhibit the D-loop activity of RAD51

Abstract RAD51 plays a central role in homologous recombination (HR), which maintains genome integrity. RAD51 is commonly overexpressed in cancer cells relative to normal tissue and is considered a therapeutic target in oncology. One potential challenge for targeting RAD51 pharmacologically is that it mediates functions in both double-strand DNA break (DSB) repair and stabilization of stalled replication forks. In order to distinguish compound-mediated effects on these RAD51 functions, we developed a novel class of RAD51 inhibitors. In contrast to many previously reported RAD51 inhibitors, we sought to develop compounds that do not inhibit RAD51's ability to bind single-stranded DNA (ssDNA). Instead, these compounds prevent RAD51-ssDNA nucleoprotein filaments from invading into homologous double-stranded DNA templates and forming D-loops. Our initial lead compound RI(dl)-1 (an abbreviation for RAD51 inhibitor of D-loop formation #1) is capable of blocking RAD51-mediated D-loop formation in biochemical assays, using concentrations that do not prevent RAD51 binding to ssDNA. An analog of this compound, termed RI(dl)-2, provides even better inhibition of RAD51's D-loop activity in biochemical systems (IC50 15.8 µM). RI(dl)-2 reduces HR activity in cells in dose ranges that do not stimulate single strand annealing (SSA) activity, which distinguishes it more generalized RAD51 inhibitors. RI(dl)-2 also sensitizes several cancer cell lines to radiation-induced death. We now present the results of more extensive structure activity relationship (SAR) optimizations aimed at further improving the potency and selectivity of this class of compounds. Several of these compounds are capable of delaying the timely resolution of radiation-induced RAD51 foci, even though initial RAD51 foci are apparently normal at earlier time points. Similar focus kinetics are observed for gamma-H2AX in identically treated cells, supporting the interpretation that these compounds permit RAD51 assembly at DSBs but prevent the completion of DSB repair. Unlike RI(dl)-2, some these newer compounds sensitize cancer cells to mitomycin C. These specialized RAD51 inhibitory compounds are attractive candidates for potential use as radiation or chemotherapy sensitizers, since they may exert fewer toxic risks compared to more generalized inhibitors of the HR repair machinery. They may also serve to help distinguish the functions of RAD51 DSB repair and replication stress tolerance, which may enable better understanding of RAD51's roles in maintaining genome stability. This abstract is also being presented as Poster A02. Citation Format: Brian Budke, Wei Lv, Werner Tueckmantel, Alan Kozikowski, Philip Connell. Small molecules that specifically inhibit the D-loop activity of RAD51 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR02.

A zinc finger nuclease assay to rapidly quantitate homologous recombination proficiency in human cell lines

Homologous recombination (HR) is a cellular mechanism for accurate repair of double-strand DNA breaks, often deregulated in cancer. Development of novel cancer therapeutics targeting HR pathways would benefit from a quantitative and rapid means of measuring HR. Here, we describe a zinc finger nuclease (ZFN) assay that can quantify HR. Knockdown of BRCA1 or inactivation of BRCA2 decreased HR activity in cells, whereas gene restoration induced activity 8-fold. HR activity was also reflective of BRCA1/2 status in cells with known endogenous mutations.