Repair of Double stranded DNA breaks-pathway choices and more
Double stranded DNA breaks (DSBs) are critical for cell health as a single unrepaired DSB is sufficient for inducing apoptosis. Two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ), have evolved to deal with DSBs and are regulated by factors that are conserved from yeast to mammals. The relative contribution of the competing DSB repair pathways differ in the different cell types and in different phases of the cell cycle, and this balance is critical for maintaining genomic stability.
Interplay of DSB repair pathways during cell cycle and impact on therapy: A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). DSB end resection must be appropriately restricted to S/G2 phases of the cell cycle, as HR requires the presence of an intact sister chromatid. Depletion of NHEJ promoting factors such as 53BP1 allows DNA end resection in the G1 phase, thereby impairing DSB repair and causing genomic instability. Conversely, loss of the HR protein, BRCA1 (critical for initiating end resection) allows the error-prone NHEJ pathway to dominate throughout the cell cycle potentially also contributing to mutations/deletions. From the perspective of therapy, loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of the DNA repair protein, poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Surprisingly, loss of 53BP1 or associated factors (RIF1, PTIP) in these tumors render them insensitive to PARP inhibitors, as DNA end resection and the subsequent steps of the HR pathway is restored.
(a) MicroRNAs in DSB repair –pathway choice and therapeutic implications: We have conducted functional screens to discover microRNA (miRNA)s that down-modulate DSB repair proteins, and influence specific repair pathways. Ectopic expression of NHEJ-regulating miRNAs in BRCA1-deficient ovarian causes resistance to PARP inhibitors and platinum-drugs. In turn these patients have poor prognosis. We now utilize focused sequencing of miRNAs from BRCA deficient tumors that are resistant to therapy to identify miRNAs that influence the DNA repair machinery. Importantly these miRNAs have significant clinical relevance as therapeutic targets or predictive biomarkers of response.
(b) Systematic identification of factors that influence the BRCA-pathway: The (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines with disrupted genes. We are utilizing the whole-genome CRISPR library to comprehensively identify factors that restore HR-mediated DSB repair in BRCA1/2-deficient ovarian tumors and make these tumors resistant to PARP inhibitors and platinum-drugs. The goal is to better understand the biology of DSB repair, cross-talk with other signaling pathways and elucidate the mechanism of chemo-resistance.
(c) Non-coding (nc) RNAs and RNA binding proteins that impact DSB repair: Using cross-linking/immunoprecipitation and RNA-Seq we now have evidence that uncharacterized non-coding RNAs may be associated with DNA repair factors such as 53BP1 and directly impact the repair process. We are investigating the precise mechanism by which these ncRNAs associate with and functionally impact DSB repair. Central to all 53BP1 activities is its recruitment to DSBs via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2).We have identified an uncharacterized RNA binding protein, TIRR (Tudor Interacting Repair Regulator) that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Over-expression of TIRR impedes 53BP1 function by blocking its localization to DSBs. Depletion of TIRR destabilizes 53BP1 in the nuclear soluble fraction and also alters the DSB-induced protein complex centering 53BP1 to broadly impede repair. We continue to characterize, TIRR as a new factor that influences DNA repair.
Down-regulation of DSB repair in mitosis and activation via dephosphorylation: Interestingly in mitosis DSBs are recognized but not repaired, that is, factors like 53BP1 and BRCA1 are excluded from DSBs. Mitosis is the only phase of the cell cycle that lacks a DNA damage checkpoint.
(a) Why activating DSB repair pathways in mitosis leads to genomic instability? We have recently observed that phosphorylation of 53BP1 at specific residues during mitosis impedes its recruitment to chromatin and DSBs. Dephosphorylation via a PP4/R3beta phosphatase complex restores activity in the G1 phase. Counter-intuitively we observe that ectopic activation of the 53BP1 pathway, consequently the NHEJ pathway, in mitotic cells causes genomic instability and mitotic defects. We are addressing this question using a combination of cytological tools and single-cell sequencing.
(b) Phosphatases in DSB repair. We have demonstrated that phosphatases participate in multiple steps of the DNA damage response, which includes facilitating DNA repair in the context of the cell cycle phase, restoration of chromatin structure and regulating checkpoints. We now have evidence that dephosphorylation of specific phospho-residues of DNA repair proteins is a pre-requisite for their function. We have developed a novel phospho-proteomic strategy to identify these factors and also to identify phosphatases that regulate their activity. Our goal is to continue these studies and systematically investigate the role of phosphatases in DSB repair.
Serum miRNAs as biomarkers
Of the various classes of circulating nucleic acids, miRNAs have been recently characterized, and are rapidly emerging as useful non-invasive biomarkers for various pathological conditions. Most of the published studies using circulating miRNAs as biomarkers have focused on cancer. Despite high concentration of RNAses in plasma and serum, circulating miRNAs are surprisingly stable and the levels are reproducibly consistent across individuals of the same species. Some of the key molecular properties of miRNAs include stability against external impacts such as enzymatic degradation, freezing, and thawing, or intense pH conditions. Several reports indicate that miRNAs are distinctly more stable than mRNAs and a modest number of miRNAs may be sufficient to serve as markers to differentiate a pathological condition. miRNAs are detected and quantitatively assayed via high-throughput expression analysis which is rapid, sensitive and, with multiple PCR-based strategies available, technically easy compared to other biomarkers.
Radiation and serum miRNAs: The exposure of human populations to radiation, either accidentally (e.g. nuclear plant incident) or intentionally (e.g. terrorism) poses a significant threat to public health. One of the most important steps in the medical management of a nuclear disaster is to triage individuals who are minimally exposed and do not need treatment compared to those who received a dose of radiation that has caused significant injury to internal organs and tissues. Existing biodosimetry techniques and devices do not predict the severity of injury sustained by specific organs and tissues, and thus do not allow for the prompt organ- and tissue-directed medical treatment that might be provided by any available radiation medical countermeasures. We began to address this question by identifying serum miRNAs that predict long-term radiation-induced hematopoietic injury in mice. Recently we used non-human primates (NHPs) to demonstrate that this concept is evolutionarily conserved and thus that serum miRNA signatures have the potential to serve as prediction biomarkers for radiation-induced fatality in a human population. Genomic analysis of these radiation-responsive conserved miRNAs revealed that a common transcriptional network regulates these miRNAs in human, mice, and NHPs. From the clinical perspective, peripheral blood cells from radiation-therapy patients also show radiation-induced changes in miRNA expression. We also observed that differential expression of serum miRNAs may reflect biological tumor response (or resistance) to thoracic radiation in non-small-cell-lung cancer (NSCLC) patients.
(a) Predicting Radiation Induced Mortality: In animal models we have evidence that distinct sets of serum miRNAs correlates with radiation dose, and also the time-frame after radiation exposure (that is days versus weeks). Overall the goal is to build on our preliminary studies and establish miRNA signatures that predict radiation injury to allow for timely and appropriate treatment of radiation victims.
(b) Radiation Therapy Induced Secondary Malignancy: While a frequently used and effective element of multimodality cancer treatment, radiation therapy results in toxicities to normal tissues that represent a significant public health burden. These risks are amplified in pediatric patients who survive their primary malignancy and carry a 20.5% 30-year cumulative risk of second malignancies, with a 2.7x relative risk conferred by radiation. 25 years after diagnosis, the death rate due to subsequent malignancies in these pediatric patients exceeds that due to all other causes. An urgent need thus exists for noninvasive biomarkers of second malignancy risk that can be evaluated early in the disease process, in time for potential interventions to reduce such risk. We hypothesize that miRNAs present in serum drawn soon after irradiation will predict underlying susceptibility to long-term radiation-induced cancers and identify pediatric patients at increased risk for this all-too-common consequence.
Early Detection of Ovarian cancer utilizing serum miRNAs: Ovarian cancer is one of the most lethal malignant gynecological tumors. Its incidence rate ranks the second among malignant tumors of the genital system following uterine corpus cancer, but its mortality rate was the highest. Given that ovarian cancer is located deep within the pelvis and is difficult to touch, as well as the lack of typical early symptoms and effective diagnostic methods, more than 70% of patients are diagnosed at advanced stage. At this stage, the tumor has metastasized into the peritoneal cavity or to distant locations which significantly limits the efficacy of therapy. Although surgical treatment and chemotherapy of ovarian cancer have improved in recent years, the prognosis of ovarian cancer remains poor. The five-year survival rate for all stages of ovarian cancer is 47%. However, for cases where a diagnosis of the disease is made early, when the cancer is still confined to the primary site, the five-year survival rate is 92.7%. Hence, there is a great need for identification of novel non-invasive biomarkers for early tumor detection. Our initial analysis indicates that a miRNA signature containing a relatively small number of targets may have high accuracy for predicting invasive ovarian cancer. Furthermore, we may be able to distinguish borderline tumors from early invasive ovarian cancers. We will continue to test the hypothesis that serum miRNA signatures may allow early detection of malignant ovarian carcinomas.