In budding yeast, Dmc1's recombinogenic activity is controlled by the meiosis-specific heterodimer Mei5-Sae3. Mei5-Sae3 is required for assembly of Dmc1 at sites of meiotic DNA double-stranded breaks. Here, we report Mei5-Sae3 can stabilize Dmc1 filaments in both the active and inactive allosteric conformations depending on the nucleotide cofactor supporting filament formation. Mei5-Sae3 specifically stabilizes the active filament form without inhibiting ATP hydrolysis, in contrast to high concentrations of calcium, AMP-PNP, and the E157D mutation in Dmc1, each of which promotes Dmc1 filament stability by processes that include blocks to ATP hydrolysis. Mei5-Sae3 increases Dmc1 ATP hydrolysis by a mechanism that could be a cause of active filament stabilization or a secondary and inconsequential effect of active filament stabilization. Mei5-Sae3 can also stabilize filaments in the inactive conformation with ADP as a cofactor. These results show that Mei5-Sae3's filament stabilization activity does not fully depend on alteration of the hydrolytic cycle. We also show Dmc1-E157D, a gain-of-function protein that bypasses the requirement for Mei5-Sae3 in vivo, is defective in ATPase activity and stabilizes the active form of Dmc1 filaments as predicted by previous observations. Hence, Dmc1's homology search and strand exchange activities do not depend on its ability to hydrolyze ATP.

pRPA32 expression in neuroblastoma cells treated with OTS167 and CPT.

MELK and EZH2 RNA expression

PDF file - 403KB, Supplemental Figure 1. Quantifications of western blots. Supplemental Figure 2. Knockdown of RAD51 in PC3 does not protect cells from the toxicity of ionizing radiation. Supplemental Table 1. Quantifications of statistical significance are displayed for the RS-1 toxicity data that are displayed in Figure 2. Supplemental Methods. Chemical synthesis of compounds.

MYCN/MYC expression in N-type and S-type neuroblastoma cell lines

Monomethoxy poly(ethylene glycol)-block-poly(trimethylene carbonate) (mPEG3–PTMC11, Mn of mPEG=3.1×103 and Mn of PTMC=10.8×103g/mol) was synthesized by ring-opening polymerization of TMC using mPEG3 as an initiator and stannous octoate as a catalyst. The block copolymer has a broad melting range with a peak at 49.5°C and a heat of fusion of 47.6J/g. The heat of fusion normalized to the mPEG content is higher than that of the mPEG3 polymer, suggesting that PTMC segments are also semi-crystalline. The mPEG3–PTMC11 films were stable in water at room temperature, whereas at 37°C the film specimens disintegrated and the amphiphilic block copolymer self-assembled into micellar-like nanoparticles with average sizes up to 210nm. The critical association concentration (CAC) of the formed micellar-like particles is 1.35×10−3mg/ml. The average size and polydispersity index of the formed mPEG3–PTMC11 nanoparticles depend on temperature and storage time: the values decrease with increases in temperature and in storage time. By co-dissolving dexamethasone with mPEG3–PTMC11 during the film preparation, micellar-like nanoparticles loaded with dexamethasone can be obtained after the film to micellar-like nanoparticles transition. A high loading efficiency of 93.3wt.% was achieved. The sustained release of the drug was complete in 20d.

NFKB2 total and nuclear protein expression increases as radiation dose increases. Knocking out NFKB2 causes radiation sensitization, suggesting that NFKB2 may be a useful target for future cancer therapies.

Homologous recombination (HR) repairs DNA double strand breaks (DSBs) and promotes tolerance of replication-blocking lesions. Cells deficient in HR are hypersensitive to radiation and DNA-damaging chemotherapies. The central recombinase protein, RAD51, is frequently overexpressed in human malignancies, thereby elevating HR efficiency and promoting resistance to DNA-damaging therapies. Our preliminary data demonstrate that non-canonical NF-κB factors control RAD51 expression, suggesting that the non-canonical NF-κB pathway may represent a therapeutic target to reverse RAD51 overexpression in tumors, rendering them susceptible to DNA-damaging treatments including radiotherapy. NFKB1, which encodes the central non-canonical NF-κB factor proteins p100/p52, was transcriptionally targeted using pools of siRNAs. Parallel experiments were performed using various human cells lines derived from a wide range of malignancies, including prostate (PC-3), osteosarcoma (U2OS), kidney (HEK-293), and colorectal carcinomas (DLD-1). Two such cell lines (U2OS and HEK-293) carry integrated reporter constructs that quantify the proficiency of HR, as well as related DSB repair pathways including single strand annealing (SSA), micro-homology mediated end joining (MMEJ), and non-homologous end joining (NHEJ). Transcriptional silencing of p100/p52 reduces RAD51 protein levels in multiple human cancer cells lines. Whole transcriptome sequencing of U2OS cells following treatment with siNFKB1 demonstrates that RAD51 is the most differentially expressed gene (4.5-fold downregulated, p<10-46) out of 40 select genes with known relevance to HR. The functional impact of p52-dependent signaling on HR proficiency was measured using the DR-GFP assay after treatment with siNFKB1. We find that siNFKB2 reduces HR proficiency by 60.6% in U2OS cells and by 58.3% in HEK-293 cells. By contrast, the proficiency of SSA (p = 0.32), MMEJ (p = 0.62), and NHEJ (p = 0.29) are unaffected by siNFKB2. Clonogenic survival assays were performed using CHO or DLD-1 cell lines that differ only in their expression of a key HR protein (XRCC3 or BRCA2, respectively). In both cell pairs, targeted disruption of NFKB2 sensitizes the HR-competent cells to either camptothecin or radiation. However, sensitization is absent in HR-incompetent control cells. The non-canonical NF-κB factor p100/p52 is a key transcriptional regulator of DNA repair proteins, most notably, the RAD51 recombinase that promotes HR. Inhibition of p100/p52-dependent signaling leads to a significant reduction HR activity in cancer cells, thereby promoting sensitization to DNA-damaging oncologic treatment.

RAD51 is the central protein in homologous recombination (HR) repair, where it first binds ssDNA and then catalyzes strand invasion via a D-loop intermediate. Additionally, RAD51 plays a role in faithful DNA replication by protecting stalled replication forks; this requires RAD51 to bind DNA but may not require the strand invasion activity of RAD51. We previously described a small-molecule inhibitor of RAD51 named RI(dl)-2 (RAD51 inhibitor of D-loop formation #2, hereafter called 2 h), which inhibits D-loop activity while sparing ssDNA binding. However, 2 h is limited in its ability to inhibit HR in vivo, preventing only about 50 % of total HR events in cells. We sought to improve upon this by performing a structure-activity relationship (SAR) campaign for more potent analogues of 2 h. Most compounds were prepared from 1-(2-aminophenyl)pyrroles by forming the quinoxaline moiety either by condensation with aldehydes, then dehydrogenation of the resulting 4,5-dihydro intermediates, or by condensation with N,N '-carbonyldiimidazole, chlorination, and installation of the 4-substituent through Suzuki-Miyaura coupling. Many analogues exhibited enhanced activity against human RAD51, but in several of these compounds the increased inhibition was due to the introduction of dsDNA intercalation activity. We developed a sensitive assay to measure dsDNA intercalation, and identified two analogues of 2 h that promote complete HR inhibition in cells while exerting minimal intercalation activity.

Abstract Maternal embryonic leucine zipper kinase (MELK) activates pathways that mediate aggressive tumor growth and therapy resistance in many types of adult cancers. Pharmacologic and genomic inhibition of MELK impairs tumor growth and increases sensitivity to radiation and chemotherapy. On the basis of these promising preclinical studies, early-phase adult clinical trials testing the MELK inhibitor OTS167 are ongoing. To investigate whether MELK is also a therapeutic target in neuroblastoma, we analyzed MELK expression in primary tumors and cell lines, and examined the effects of OTS167 on neuroblastoma growth. In primary tumors, high levels of MELK were associated with advanced stage disease and inferior survival. Higher levels of MELK were also detected in tumorigenic versus nontumorigenic neuroblastoma cell lines, and cells with higher levels of MELK expression were more sensitive to OTS167 than low-MELK expressing cells. OTS167 suppressed the growth of neuroblastoma xenografts, and in a preclinical model of minimal residual disease, survival was prolonged with MELK inhibition. OTS167 treatment downregulated MELK and its target enhancer of zeste homolog 2 (EZH2), a component of the polycomb repressive complex 2 (PRC2) that is known to modulate the DNA damage response. We also show that OTS167 reduced the formation of collapsed replication forks induced by camptothecin or radiation. Taken together, our results indicate that MELK indirectly mediates efficient processing of replication-associated DNA lesions in neuroblastoma, and that OTS167 sensitizes cells to DNA-damaging agents by abrogating this process. Further studies evaluating the activity of combination treatment regimens with OTS167 in neuroblastoma are warranted.

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.

Homologous recombination (HR) is an evolutionarily conserved DNA repair process. Overexpression of the key HR protein RAD51 is a common feature of malignant cells. RAD51 plays two distinct genome-stabilizing roles, including HR-mediated repair of double-strand breaks (DSBs) and the promotion of replication fork stability during replication stress. Because upregulation of RAD51 in cancer cells can promote tumor resistance to DNA-damaging oncologic therapies, we and others have worked to develop cancer therapeutics that target various aspects of RAD51 protein function. Herein, we provide an overview of recent developments in this field, together with our perspectives on the challenges associated with these evolving anticancer strategies.

RAD51 is the central protein in homologous recombination (HR) DNA repair and represents a therapeutic target in oncology. Herein we report a novel class of RAD51 inhibitors that were identified by high throughput screening. In contrast to many previously reported RAD51 inhibitors, our lead compound 1 is capable of blocking RAD51-mediated D-loop formation (IC50 21.3 ± 7.8 μM) at concentrations that do not influence RAD51 binding to ssDNA. In human cells, 1 inhibits HR (IC50 13.1 ± 1.6 μM) without blocking RAD51's ability to assemble into subnuclear foci at sites of DNA damage. We determined that the active constituent of 1 is actually an oxidized derivative (termed RI(dl)-1 or 8) of the original screening compound. Our SAR campaign also yielded RI(dl)-2 (hereafter termed 9h), which effectively blocks RAD51's D-loop activity in biochemical systems (IC50 11.1 ± 1.3 μM) and inhibits HR activity in human cells (IC50 3.0 ± 1.8 μM).

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.

Mutagenesis is a hallmark of malignancy, and many oncologic treatments function by generating additional DNA damage. Therefore, DNA damage repair is centrally important in both carcinogenesis and cancer treatment. Homologous recombination (HR) and nonhomologous end joining are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The recombination proficiency score (RPS) is based on the expression levels for four genes involved in DNA repair pathway preference (Rif1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLCs). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression contributes to the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteract the adverse prognosis associated with low RPS. Therefore, RPS may help oncologists select which therapies will be effective for individual patients, thereby enabling more personalized care.

Abstract RAD51 is the central protein that catalyzes DNA repair via homologous recombination, a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their noncancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA-binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from dsDNA. In PC3 prostate cancer cells, RS-1–induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant antitumor responses in a mouse model, providing proof-of-principle for this novel therapeutic strategy. Cancer Res; 74(13); 3546–55. ©2014 AACR.

RAD51 is the central strand exchange recombinase in somatic homologous recombination, providing genomic stability and promoting resistance to DNA damage. An important tool for mechanistic studies of RAD51 is the D-loop or strand assimilation assay, which measures the ability of RAD51-coated single-stranded DNA (ssDNA) to search for, invade and exchange ssDNA strands with a homologous duplex DNA target. As cancer cells generally overexpress RAD51, the D-loop assay has also emerged as an important tool in oncologic drug design programs for targeting RAD51. Previous studies have adapted the traditional gel-based D-loop assay by using fluorescence-based substrates, which in principle allow for use in high-throughput screening platforms. However, these existing D-loop methods depend on linear oligonucleotide DNA duplex targets, and these substrates enable recombinase-independent ssDNA annealing that can obscure the recombinase-dependent strand assimilation signal. This compelled us to fundamentally re-design this assay, using a fluorescent target substrate that consists of a covalently closed linear double-hairpin dsDNA. This new microplate-based method represents a fast, inexpensive and non-radioactive alternative to existing D-loop assays. It provides accurate kinetic analysis of strand assimilation in high-throughput and performs well with human RAD51 and Escherichia coli RecA protein. This advance will aid in both mechanistic studies of homologous recombination and drug screening programs.