2015 — 2016 |
Bindra, Ranjit |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Development of a Novel Assay to Measure Dsb Repair At Endogenous Loci in Cells
? DESCRIPTION (provided by applicant): The goal of this R03 application is to develop a novel method to measure DSB repair at endogenous loci, using a next-generation sequencing platform and automated alignment program that was recently developed in our laboratory. This method is unique from previously described assays, because it does not require the integration of artificial sequences into the genome, and it does not rely on a fluorescent protein as a reporter. As such, our assay could be rapidly applied to many different cell lines, and even primary tumor cultures in the future. Thus, our approach will have uses for both basic science and clinical applications. DSBs are a serious threat to genomic integrity and cell survival, and thus complex pathways have evolved which can rapidly detect DNA breaks, activate cell cycle checkpoints to arrest growth, and then repair these lesions. In addition, tumor cell survival often is dependent on the repair of DSBs induced by anti-cancer treatments, such as ionizing radiation (IR) and many chemotherapeutics. As such, there has been intense interest in elucidating the key proteins involved in DSB repair, and the mechanisms by which they function. Such insights would be important for a better understanding of the DNA damage response in mammalian cells and in developing novel cancer therapeutics. There are two key DSB repair pathways in mammalian cells, non- homologous end joining (NHEJ) and homologous recombination (HR). There have been numerous insights into the key players in these pathways, and even recently many new proteins were found to play important roles in the recognition, triage and repair of DSBs. DSB repair assays are critical for these research efforts, and better assays are needed which report on repair events that occur at endogenous sites in the genome. I started my laboratory in August of 2012, and during our first 2 years we developed a novel approach to assess DSB repair at genomic loci using next-generation sequencing and automated sequence alignment. In preliminary studies, we created a next-generation sequencing protocol and we demonstrated initial feasibility with an engineered nuclease site in cells. In Aim 1, we will adapt our platform to measure NHEJ at multiple endogenous genomic sites. In Aim 2, we will further engineer our system to measure both NHEJ and HR at a naturally occurring region of flanking sequence homology in the genome. We are proposing to develop a completely novel method to measure DSB repair, which does not require the integration of sequences into the genome, and which does not rely on a fluorescent protein as a reporter. The lack of dependence on a fluorescent protein as a read-out means that DSB repair activity can be assessed nearly instantaneously, and at multiple time-points with fine resolution. The experiments proposed in this R03 will serve as a critical proof-of-principle that both mutagenic NHEJ and HR can be measured at endogenous loci using a next-generation sequencing-based approach. Thus, they are consistent with the R03 grant mechanism which supports the development of new research technology and methodology.
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0.915 |
2017 — 2018 |
Bindra, Ranjit Saltzman, W Mark [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Drug Delivery System For Enhancing Radiation Therapy in Pediatric Glioma
PROJECT SUMMARY Pediatric high-grade gliomas are devastating central nervous system (CNS) tumors. In the most malignant form of the disease, glioblastoma multiforme (GBM), over 80% of children will succumb to their disease within 5 years after surgery, radiation therapy (RT) and chemotherapy. Many systemic agents have been tested over the last 40 years without changes in survival. These agents likely failed because they cannot cross the blood- brain-barrier (BBB). For the minority of drugs with CNS penetration, high concentrations and uniform distributions in the brain remain a major efficacy barrier. Attempts at systemic dose escalation are limited by extracranial toxicity. As such, better formulations and drug delivery systems are needed for this disease. Such formulations have been created and tested for adult brain tumors, but their application in the pediatric setting has been lacking. To address this unmet need, we propose a multi-PI investigator approach?led by a bioengineer and an oncology physician/scientist?to develop a novel approach for safe and effective drug delivery in the brain for treating pediatric tumors. Although the BBB limits the effectiveness of chemotherapy in pediatric brain tumors, it is now known that agents can be delivered locally?directly into the brain, beyond the blood-brain barrier. In adults, this is accomplished with a dime-size degradable wafer (Gliadel®) that is placed in the tumor resection bed during surgery. Gliadel® slowly releases the drug for several weeks after placement. But Gliadel® has limitations. Because it relies on diffusion of an agent into the tissue, high levels of drug are achieved only within a few millimeters of the wafer. Areas of tumor infiltration in the surrounding brain may be several centimeters from the wafer, and the drugs likely cannot reach them. Another approach, called convection-enhanced delivery (CED), potentially solves these problems by using fluid flow to carry drug molecules through the brain. CED has been shown to be safe but, so far, CED treatments have not been effective. Part of the problem is that the infusion of agents dissolved in liquid, even by CED, does not provide the necessary duration of exposure. We recognize these limitations and are creating an improved drug formulation/delivery system in which 1) nanoparticles are infused locally into the tumor by CED; 2) the nanoparticles are loaded with DNA repair inhibitors known to chemo- and radio-sensitize gliomas; and 3) the nanoparticles release the inhibitors over a sustained time period, so that the agents are present in tumor cells throughout the course of conformal RT, and even during adjuvant chemotherapy. In this unprecedented triple targeted approach, nanoparticles gain access to targeted regions of the brain, releasing agents that target DNA repair in the tumor, and RT is targeted to the volume of the brain where the tumor cells reside. Given that the nanoparticles are composed of a polymer that is approved by the FDA for human use, this approach has the potential to be rapidly adopted into practice, and it could radically change the way that pediatric GBMs are treated.
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0.915 |
2017 — 2021 |
Bindra, Ranjit |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Exploiting Mutant Idh1/2-Induced Homologous Recombination Defects in Cancer
Only a handful of therapies are available for treatment in the front-line for glioma: radiotherapy, temozolomide, and PCV (procarbazine, CCNU, and vincristine). These therapies have not changed in over 3 decades, and in most cases they are not curative, nor are they targeted to the underlying mutations driving these tumors. 2-Hydroxyglutarate (2HG) exists as two enantiomers, R-2HG and S-2HG, and both are implicated in tumor progression via their inhibitory effects on ?-ketoglutarate (?KG)-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. Our laboratory recently discovered 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 inhibitors, and it can be entirely recapitulated by treatment with either 2HG enantiomer in cells with intact IDH1/2. We demonstrated IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in vitro and genetically-matched tumor xenografts in vivo. These findings directly challenge the current therapeutic strategy to block IDH1/2 mutant function by direct inhibition, and they instead provide a novel approach to treat these tumors with DNA repair inhibitors. Furthermore, our results uncover an unexpected link between oncometabolites, DNA repair and genetic instability. Based on the preliminary data presented above, our central hypothesis is that IDH1/2-mutant tumors harbor intrinsic double-strand break (DSB) repair defects, which can be exploited for a therapeutic gain. The overall goals of this application are (1) to understand how R-2HG and related oncometabolites, which are induced by IDH1/2 mutations and other processes, suppress DSB repair, (2) how DSB repair specifically is affected by these oncometabolites, and (3) the most effective way to exploit this defect using DNA repair inhibitor-based treatment regimens. In Aim 1 of this application, we will test the hypothesis that specific ?KG- dependent dioxygenases mediate the observed phenotype of oncometabolite-induced DSB repair suppression. In Aim 2, we will perform a comprehensive evaluation of key nodes in the DNA damage response network, in order to localize the exact mechanism(s) of action by which DSB repair is suppressed. Finally, in Aim 3, we will test the hypothesis that the oncometabolite-induced DSB repair defect can be targeted by combination treatment with DNA repair inhibitors and DNA damaging agents. The long term goal of this study is to translate our novel findings into a clinical trial, in which we will test the efficacy of the combination therapies that are identified in this proposal. This trial would be tissue-agnostic and biomarker-driven, focusing on tumors that produce high levels of oncometabolites, which can be exploited with DNA repair inhibitor-based regimens.
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0.915 |