Double-strand Break Homologous Recombination Research Articles

Precision Medicine for BRCA/PALB2-Mutated Pancreatic Cancer and Emerging Strategies to Improve Therapeutic Responses to PARP Inhibition.

Simple SummaryFor the small subset of pancreatic ductal adenocarcinoma (PDAC) patients with loss-of-function mutations to BRCA1/2 or PALB2, both first-line and maintenance therapy differs significantly. These mutations confer a loss of double-strand break DNA homologous recombination (HR), substantially altering drug sensitivities. In this review, we discuss the current treatment guidelines for PDAC tumors deficient in HR, as well as newly emerging strategies to improve drug responses in this population. We also highlight additional patient populations in which these strategies may also be effective, and novel strategies aiming to confer similar drug sensitivity to tumors proficient in HR repair.Pancreatic cancer is projected to become the second leading cause of cancer-related death by 2030. As patients typically present with advanced disease and show poor responses to broad-spectrum chemotherapy, overall survival remains a dismal 10%. This underscores an urgent clinical need to identify new therapeutic approaches for PDAC patients. Precision medicine is now the standard of care for several difficult-to-treat cancer histologies. Such approaches involve the identification of a clinically actionable molecular feature, which is matched to an appropriate targeted therapy. Selective poly (ADP-ribose) polymerase (PARP) inhibitors such as Niraparib, Olaparib, Talazoparib, Rucaparib, and Veliparib are now approved for several cancers with loss of high-fidelity double-strand break homologous recombination (HR), namely those with deleterious mutations to BRCA1/2, PALB2, and other functionally related genes. Recent evidence suggests that the presence of such mutations in pancreatic ductal adenocarcinoma (PDAC), the most common and lethal pancreatic cancer histotype, significantly alters drug responses both with respect to first-line chemotherapy and maintenance therapy. In this review, we discuss the current treatment paradigm for PDAC tumors with confirmed deficits in double-strand break HR, as well as emerging strategies to both improve responses to PARP inhibition in HR-deficient PDAC and confer sensitivity to tumors proficient in HR repair.

Comprehensive genomic analysis of refractory multiple myeloma reveals a complex mutational landscape associated with drug resistance and novel therapeutic vulnerabilities

The outcomes of patients with multiple myeloma (MM) refractory to immunomodulatory agents (IMiDs) and proteasome inhibitors (PIs) remain poor. In this study, we performed whole genome and transcriptome sequencing of 39 heavily pretreated relapsed/refractory MM (RRMM) patients to identify mechanisms of resistance and potential therapeutic targets. We observed a high mutational load and indications of increased genomic instability. Recurrently mutated genes in RRMM, which had not been previously reported or only observed at a lower frequency in newly diagnosed MM, included NRAS, BRAF, TP53, SLC4A7, MLLT4, EWSR1, HCFC2, and COPS3. We found multiple genomic regions with bi-allelic events affecting tumor suppressor genes and demonstrated a significant adverse impact of bi-allelic TP53 alterations on survival. With regard to potentially resistance conferring mutations, recurrently mutated gene networks included genes with relevance for PI and IMiD activity; the latter particularly affecting members of the Cereblon and the COP9 signalosome complex. We observed a major impact of signatures associated with exposure to melphalan or impaired DNA double-strand break homologous recombination repair in RRMM. The latter coincided with mutations in genes associated with PARP inhibitor sensitivity in 49% of RRMM patients; a finding with potential therapeutic implications. In conclusion, this comprehensive genomic characterization revealed a complex mutational and structural landscape in RRMM and highlights potential implications for therapeutic strategies.

Frequency and prognostic value of mutations associated with the homologous recombination DNA repair pathway in a large pan cancer cohort

PARP inhibitors have shown remarkable efficacy in the clinical management of several BRCA-mutated tumors. This approach is based on the long-standing hypothesis that PARP inhibition will impair the repair of single stranded breaks, causing synthetic lethality in tumors with loss of high-fidelity double-strand break homologous recombination. While this is now well accepted and has been the basis of several successful clinical trials, emerging evidence strongly suggests that mutation to several additional genes involved in homologous recombination may also have predictive value for PARP inhibitors. While this notion is supported by early clinical evidence, the mutation frequencies of these and other functionally related genes are largely unknown, particularly in cancers not classically associated with homologous recombination deficiency. We therefore evaluated the mutation status of 22 genes associated with the homologous recombination DNA repair pathway or PARP inhibitor sensitivity, first in a pan-cancer cohort of 55,586 patients, followed by a more focused analysis in The Cancer Genome Atlas cohort of 12,153 patients. In both groups we observed high rates of mutations in a variety of HR-associated genes largely unexplored in the setting of PARP inhibition, many of which were associated also with poor clinical outcomes. We then extended our study to determine which mutations have a known oncogenic role, as well as similar to known oncogenic mutations that may have a similar phenotype. Finally, we explored the individual cancer histologies in which these genomic alterations are most frequent. We concluded that the rates of deleterious mutations affecting genes associated with the homologous recombination pathway may be underrepresented in a wide range of human cancers, and several of these genes warrant further and more focused investigation, particularly in the setting of PARP inhibition and HR deficiency.

Abstract 2565: Type I interferons enhance chemosensitivity of breast cancer by inhibiting DNA repair

Abstract Type I interferons (IFNs) are known to have antineoplastic effects against several tumors by stimulating anticancer immune response. In this study, we identified a novel function of type I IFNs in DNA repair, which enhanced chemosensitivity in breast cancer cells. We investigated that IFNα and IFNβ inhibited both non-homologous recombination and homologous recombination double strand breaks repair. Moreover, IFNs accelerated the release of DNA into the cytoplasm in response to DNA damaging agent, doxorubicin. In agreement, IFNs synergistically activated DNA damage-induced STING pathway and increased the production of chemokines, CCL5 and CXCL11. In agreement, IFNβ greatly increased the sensitivity of cells to doxorubicin-induced suppression of cell survival. This sensitizing effect of IFNβ was also supported by in vivo xenograft experiments. Thus, combination therapy with chemotherapeutic agents and IFNs may be effective against breast cancer, particularly in patients with defective in DNA damage response pathways. The downstream effectors that modulate the effect of IFNs should be investigated as predictive biomarker in the treatment of breast cancer. Citation Format: Na-Lee Ka, Ga Young Lim, Mi-Ock Lee. Type I interferons enhance chemosensitivity of breast cancer by inhibiting DNA repair [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 2565.

Cellular Assays to Study the Functional Importance of Human DNA Repair Helicases.

DNA helicases represent a specialized class of enzymes that play crucial roles in the DNA damage response. Using the energy of nucleoside triphosphate binding and hydrolysis, helicases behave as molecular motors capable of efficiently disrupting the many noncovalent hydrogen bonds that stabilize DNA molecules with secondary structure. In addition to their importance in DNA damage sensing and signaling, DNA helicases facilitate specific steps in DNA repair mechanisms that require polynucleotide tract unwinding or resolution. Because they play fundamental roles in the DNA damage response and DNA repair, defects in helicases disrupt cellular homeostasis. Thus, helicase deficiency or inhibition may result in reduced cell proliferation and survival, apoptosis, DNA damage induction, defective localization of repair proteins to sites of genomic DNA damage, chromosomal instability, and defective DNA repair pathways such as homologous recombination of double-strand breaks. In this chapter, we will describe step-by-step protocols to assay the functional importance of human DNA repair helicases in genome stability and cellular homeostasis.

PO-147 Pinostilbene hydrate suppresses human oral cancer cell metastasis via downregulation matrix metalloproteinase-2 through the mitogen-activated protein kinase signalling pathway

IntroductionAcute myeloid leukaemia (AML) is the most common acute leukaemia in adults and has one of the poorest survival rates of all cancers. Despite advancements in treatments, the 5 year survival rate for AML remains at 26%, and is less than 8% for patients aged over 60. Approximately one-third of patients exhibit primary refractory disease, however, the mechanisms controlling this have not been fully elucidated. Overexpression of the brain and acute leukaemia, cytoplasmic (BAALC) gene is a negative prognostic factor for AML and is associated with primary refractory disease, but precisely how overexpression of BAALC is controlling sensitivity to chemotherapeutics is unknown. As AML cells with perturbations in DNA repair pathways exhibit alterations in sensitivity/resistance to a range of chemotherapeutics, we examined whether BAALC overexpression perturbs DNA repair pathways in AML cells.Material and methodsBAALC was overexpressed in a panel of AML cells and their ability to repair DNA damage was measured by western blot and comet assay. Alterations in expression of key DNA damage repair genes were measured by PCR in BAALC overexpressing and empty vector (EV) control cells. BAALC-mediated signalling complexes involved in DNA repair were identified by immunoprecipitation.Results and discussionsBAALC, but not EV, overexpression resulted in elevated DNA fragmentation (comet assay), and sustained gH2AX and ATM activation (pSer1981) post-daunorubicin and etoposide-induced DNA damage, indicative of an inability to repair the damage. Gene expression analyses in BAALC overexpressing cells identified that the expression of components (RAD51D, TP53BP1, BTG2) of the high-fidelity homologous recombination double strand break repair pathway were significantly decreased. Furthermore, BAALC interacts with several proteins (RAD51, CaMKII, dermcidin) implicated in DNA repair. Further supporting a role for BAALC overexpression in inducing defective homologous recombination repair, BAALC overexpressing AML cells were hypersensitive to cisplatin treatment.ConclusionAs perturbations in DNA repair pathways in AML have been associated with increased resistance to a range of chemotherapeutics, and BAALC overexpression is associated with primary refractory AML, our data suggests that BAALC overexpression induces decreased chemosensitivity by perturbing DNA repair pathways.

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

Homing endonucleases: from basics to therapeutic applications.

Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12-40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.

HSSB1 and hSSB2 Form Similar Multiprotein Complexes That Participate in DNA Damage Response

hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

Opposing Role of Condensin and Radiation-sensitive Gene RAD52 in Ribosomal DNA Stability Regulation

Blocking target of rapamycin signaling by starvation or rapamycin inhibits ribosomal DNA (rDNA) transcription and causes condensin-mediated rDNA condensation and nucleolar contraction. In the absence of condensin, however, repression of rDNA transcription leads to rDNA instability and elevated level of extrachromosomal rDNA circles and nucleolar fragmentation. Here, we show that mutations in the Rad52 homologous recombination machinery block rDNA instability. Rad52 is normally excluded from the nucleolus. In the absence of condensin, however, repression of rDNA transcription results in Rad52 localization to the nucleolus, association with rDNA and subsequent formation of extrachromosomal rDNA circles, and reduced cell survival. In contrast, deletion of RAD52 restores cell viability under the same conditions. These results reveal an opposing role of condensin and Rad52 in the control of rDNA stability under nutrient starvation conditions.

Combining PCI-24781, a Novel Histone Deacetylase Inhibitor, with Chemotherapy for the Treatment of Soft Tissue Sarcoma

Histone deactylase inhibitors (HDACi) are a promising new class of anticancer therapeutics; however, little is known about HDACi activity in soft tissue sarcoma (STS), a heterogeneous cohort of mesenchymal origin malignancies. Consequently, we investigated the novel HDACi PCI-24781, alone/in combination with conventional chemotherapy, to determine its potential anti-STS-related effects and the underlying mechanisms involved. Immunoblotting was used to evaluate the effects of PCI-24781 on histone and nonhistone protein acetylation and expression of potential downstream targets. Cell culture-based assays were utilized to assess the effects of PCI-24781 on STS cell growth, cell cycle progression, apoptosis, and chemosensitivity. Quantitative reverse transcription-PCR, chromatin immunoprecipitation, and reporter assays helped elucidate molecular mechanisms resulting in PCI-24781-induced Rad51 repression. The effect of PCI-24781, alone or with chemotherapy, on tumor and metastatic growth was tested in vivo using human STS xenograft models. PCI-24781 exhibited significant anti-STS proliferative activity in vitro, inducing S phase depletion, G(2)/M cell cycle arrest, and increasing apoptosis. Superior effects were seen when combined with chemotherapy. A PCI-24781-induced reduction in Rad51, a major mediator of DNA double-strand break homologous recombination repair, was shown and may be a mechanism underlying PCI-24781 chemosensitization. We showed that PCI-24781 transcriptionally represses Rad51 through an E2F binding-site on the Rad51 proximal promoter. Although single-agent PCI-24781 had modest effects on STS growth and metastasis, marked inhibition was observed when combined with chemotherapy. In light of these findings, this novel molecular-based combination may be applicable to multiple STS histologic subtypes, and potentially merits rigorous evaluation in human STS clinical trials.

Polymorphisms inRAD51, XRCC2, andXRCC3Are Not Related to Breast Cancer Risk

Highly penetrant, but rare, mutations in genes involved in double-strand break repair (i.e., BRCA1 and BRCA2 ) are associated with a risk for breast cancer of 40% to 65% by age 70 years ([1][1], [2][2]). Polymorphisms in other double-strand break repair genes are thought to contribute to the risk

Expression of the DNA Repair Gene RAD51 Is Associated with FLT3 ITD Transcript Level and Is Down-Regulated by PKC412 Resulting in Suppression of DNA Repair.

The presence of internal tandem duplication (ITD) mutations in the FLT3 receptor tyrosine kinase confer an adverse prognosis in AML due to an increased risk of disease relapse. However the mechanisms underlying this increased relapse risk are unclear. We have investigated whether AML cells with FLT3 ITD mutations have an enhanced capacity for DNA repair following cytotoxic drug exposure. RAD51 is a key protein in the high-fidelity homologous recombination double strand break repair pathway and is the limiting factor for this pathway in mammalian cells. Using quantitative real-time PCR we found that the level of RAD51 transcripts are significantly correlated with the level of FLT3 transcripts in FLT3 ITD cells (n=27; p=0.017) but not in FLT3 WT cells (n=57; p=0.58). Clinically FLT3 ITDs have the most significant prognostic impact in AML patients with normal cytogenetics and if this group is studied the association between FLT3 ITD and RAD51 transcript levels are particularly pronounced (n=12; p=0.003).To establish whether increases in RAD51 expression correlate with enhanced DNA repair activity we have adopted the comet assay and studied the MV4-11 cell line (FLT3 ITD), HL60 cell line (FLT3 WT) and a number of AML patient cells. The background level of DNA damage in untreated FLT3 ITD AML patients and the MV4-11 cell line was significantly lower than in FLT3 WT patients and the HL60 cell line (n=10; p=0.02), suggesting a constitutive up-regulation of DNA repair in cells harbouring the FLT3 ITD mutation resulting in lower background levels of DNA damage.To test whether the increase in RAD51 and DNA repair was a consequence of the FLT3 ITD, we treated cells with the FLT3 inhibitor PKC412 and then examined the response of the cells to sub-toxic doses of daunorubicin. The MV4-11 FLT3 ITD cells, but not the FLT3 WT HL60 cells, demonstrated a statistically significant suppression of early DNA repair when treated with PKC412 and daunorubicin (p<0.001). Similar results were obtained in primary AML cells, with a loss of early DNA repair in the FLT3 ITD cells and no effect on the WT FLT3 cells. Furthermore the reduction in daunorubicin-induced DNA repair seen in PKC412 treated FLT3 ITD cells was associated with down-regulation of RAD51 expression. There was a statistically significant decrease in RAD51 transcript level following PKC412 treatment in the FLT3 ITD patients and MV4-11 cell line but not in the FLT3 WT patients or HL60 cell line (p<0.05). The decrease in the expression of RAD51 is closely associated with the reduction in DNA repair function in the inhibitor treated FLT3 ITD cells.This work suggests that high expression levels of FLT3 transcripts in AML cells with a FLT3 ITD up-regulate RAD51 resulting in more efficient DNA repair following chemotherapy treatment. This may lead to resistance to cytotoxic therapies and genomic instability, ultimately resulting in the manifestation of a more resistant disease and a greater likelihood of relapse. The use of FLT3 inhibitors concurrently with or following AML therapy may suppress enhanced DNA repair in FLT3 mutated cells.

A single-nucleotide polymorphism in the RAD51 gene modifies breast cancer risk in BRCA2 carriers, but not in BRCA1 carriers or noncarriers.

Variation in the penetrance estimates for BRCA1 and BRCA2 mutation carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. The RAD51 gene, which participates in homologous recombination double-strand breaks (DSB) repair in the same pathway as the BRCA1 and BRCA2 gene products, is a candidate for such an effect. A single-nucleotide polymorphism (SNP), RAD51-135g → c, in the 5′ untranslated region of the gene has been found to elevate breast cancer (BC) risk among BRCA2 carriers. We genotyped 309 BRCA1/2 mutation carriers, of which 280 were of Ashkenazi origin, 166 noncarrier BC patients and 152 women unaffected with BC (a control group), for the RAD51-135g → c SNP. Risk analyses were conducted using COX proportional hazard models for the BRCA1/2 carriers and simple logistic regression analysis for the noncarrier case–control population. BRCA2 carriers were also studied using logistic regression and Kaplan–Meier survival analyses. The estimated BC hazard ratio (HR) for RAD51-135c carriers adjusted for origin (Ashkenazi vs non-Ashkenazi) was 1.28 (95% CI 0.85–1.90, P=0.23) for BRCA1/2 carriers, and 2.09 (95% CI 1.04–4.18, P=0.04) when the analysis was restricted to BRCA2 carriers. The median BC age was younger in BCRA2-RAD51-135c carriers (45 (95% CI 36–54) vs 52 years (95% CI 48–56), P=0.05). In a logistic regression analysis, the odds ratio (OR) was 5.49 (95% CI 0.5–58.8, P=0.163). In noncarrier BC cases, carrying RAD51-135c was not associated with BC risk (0.97; 95% CI 0.47–2.00). These results indicate significantly elevated risk for BC in carriers of BRCA2 mutations who also carry a RAD51-135c allele. In BRCA1 carriers and noncarriers, no effect for this SNP was found.