Peptide nucleic acids (PNAs) can target and stimulate recombination reactions in genomic DNA. We have reported that γPNA oligomers possessing the diethylene glycol γ-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve γPNA accessibility, we explore the utility of γPNA oligomers possessing the hydroxymethyl-γ moiety for gene-editing applications. We demonstrate that a γPNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity than that of the diethylene glycol-γPNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-γPNA stimulates higher frequencies (≥1.5-fold) of gene modification than the diethylene glycol γPNA in mouse bone marrow cells.
Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the Pms2 or Mlh1 MMR gene develop cancer but exhibit very different tumor spectra; only Mlh1 −/− animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an in vivo forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in Mlh1 −/− mice was 2- to 3-fold higher than in Pms2 −/− animals. The higher mutation frequency in Mlh1 −/− mice may be a consequence of some residual DNA repair capacity in the Pms2 −/− animals. Relevant to this idea, we observed that Pms2 −/− mice exhibit almost normal levels of Mlh1p, whereas Mlh1 −/− animals lack both Mlh1p and Pms2p. Comparison between Mlh1 −/− animals and Mlh1 −/− and Pms2 −/− double knockout mice revealed little difference in mutator phenotype, suggesting that Mlh1 nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of Mlh1 −/− mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.
Gibson, S. L., Bindra, R. S. and Glazer, P. M. CHK2-Dependent Phosphorylation of BRCA1 in Hypoxia. Radiat. Res. 166, 646–651 (2006).Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.
Tumors with neomorphic mutations in IDH1/2 have defective homologous recombination repair, resulting in sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. The Olaparib Combination trial is a phase II, open-label study in which patients with solid tumors harboring IDH1/2 mutations were treated with olaparib as monotherapy, with objective response and clinical benefit rates as the primary end points.Ten patients with IDH1/2-mutant tumors by next-generation sequencing were treated with olaparib 300 mg twice daily.Three of five patients with chondrosarcomas had clinical benefit, including one patient with a partial response and two with stable disease lasting > 7 months. A patient with pulmonary epithelioid hemangioendothelioma had stable disease lasting 11 months. In contrast, clinical benefit was not observed among four patients with cholangiocarcinoma.These results indicate preliminary activity of PARP inhibition in patients with IDH1/2-mutant chondrosarcoma and pulmonary epithelioid hemangioendothelioma. Further studies of PARP inhibitors alone and in combination in this patient population are warranted.
Abstract 2-Hydroxyglutarate (2HG) exists as two enantiomers, R-2HG and S-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, while the latter is produced under pathologic process such as hypoxia. Recurring IDH1/2 mutations were first identified gliomas and acute myeloid leukemia (AML), and subsequently they were found in multiple other tumor types. Many IDH1/2-mutant tumors are known to be chemo- and radiosensitive, although the mechanisms underlying this enhanced sensitivity have been elusive. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect which renders tumor cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. Remarkably, this “BRCAness” phenotype can be completely reversed by small molecule mutant IDH1/2 inhibitors, and it can be entirely recapitulated by treatment with either 2HG enantiomer in cells with intact IDH1/2. We performed a comprehensive series of studies directly implicate two αKG-dependent dioxygenases, KDM4A and KDM4B, as key mediators of the observed phenotype. In addition, we demonstrate that 2HG-induced HR suppression cannot be explained by mutant IDH1/2-associated alterations in NAD+ levels. We have demonstrated IDH1/2-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells and AML bone marrow cultures in vitro, as well as genetically-matched tumor xenografts in vivo. Finally, we have extended these findings to several structurally related and clinically relevant oncometabolites. We demonstrate profound synthetic lethality with PARP inhibitors in tumors which produce these other oncometabolites, and our data suggest a similar mechanism of action via which HR is suppressed. Small molecule inhibition of oncogenic kinases is a pillar of precision medicine in modern oncology, and this approach has been extrapolated to treat IDH1/2-mutant and other oncometabolite-producing cancers with small molecule inhibitors which block the neomorphic activity of the mutant proteins. The findings present here directly challenge this therapeutic strategy, and they instead provide a novel approach to treat these tumors oncometabolite-producing tumors with DNA repair inhibitors. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair and genetic instability. We previously reported that hypoxia suppresses HR, driving genetic instability and conferring a BRCAness phenotype in hypoxic tumor cells. It is tempting to speculate that the findings reported here provide a novel commonality between hypoxia and IDH1/2 mutations as mediating a “hit-and-run” mechanism for genetic instability and tumor progression through 2HG, but at the same time bestowing a vulnerability to PARP inhibition that can be therapeutically exploited. Based on these findings, we are planning a multi-center Phase II trial testing the efficacy of olaparib for the treatment of recurrent IDH1/2-mutant tumors, and we anticipate this trial will be open for enrollment later this year. Citation Format: Parker Sulkowski, Christopher Corso, Nathaniel Robinson, Susan Scanlon, Karin Purshouse, Hanwen Bai, Yanfeng Liu, Ranjini Sundaram, Denise Hegan, Nathan Fons, Gregory Breuer, Yuanbin Song, Ketu Mishra, Henk De Feyter, Robin de Graaf, Yulia Surovtseva, Maureen Kachman, Stephanie Halene, Murat Gunel, Peter Glazer, Ranjit S. Bindra. Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2017-LB-290
The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.
RAD51, a key factor in homology-directed repair (HDR), has long been considered an attractive target for cancer therapy, but few specific inhibitors have been found. A cell-penetrating, anti-DNA, lupus autoantibody, 3E10, was previously shown to inhibit HDR, sensitize tumors to radiation, and mediate synthetic lethal killing of BRCA2-deficient cancer cells, effects that were initially attributed to its affinity for DNA. However, as the molecular basis for its ability to inhibit DNA repair, we report that 3E10 directly binds to the N-terminus of RAD51, sequesters RAD51 in the cytoplasm, and impedes RAD51 binding to DNA. Further, we generate separation-of-function mutations in the complementarity-determining regions of 3E10 revealing that inhibition of HDR tracks with binding to RAD51 but not to DNA, whereas cell penetration is linked to DNA binding. The consequences of these mutations on putative 3E10 interactions with RAD51 and DNA are correlated with in silico molecular modeling. Taken together, the results identify 3E10 as a novel inhibitor of RAD51 by direct binding, accounting for its ability to suppress HDR and providing the molecular basis to guide pre-clinical development of 3E10 as an anti-cancer agent.
