RESUMO
Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.
Assuntos
Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/uso terapêuticoRESUMO
Small-molecule inhibitors of DNA repair pathways are being intensively investigated as primary and adjuvant chemotherapies. We report the discovery that cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, are potent inhibitors of DNA double-strand break (DSB) repair. Our data suggest that cardiac glycosides interact with phosphorylated mediator of DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin-protein ligase ring finger protein 8 (RNF8), two factors involved in DSB repair, and inhibit the retention of p53 binding protein 1 (53BP1) at the site of DSBs. These observations provide an explanation for the anticancer activity of this class of compounds, which has remained poorly understood for decades, and provide guidance for their clinical applications. This discovery was enabled by the development of the first high-throughput unbiased cellular assay to identify new small-molecule inhibitors of DSB repair. Our assay is based on the fully automated, time-resolved quantification of phospho-SER139-H2AX (γH2AX) and 53BP1 foci, two factors involved in the DNA damage response network, in cells treated with small molecules and ionizing radiation (IR). This primary assay is supplemented by robust secondary assays that establish lead compound potencies and provide further insights into their mechanisms of action. Although the cardiac glycosides were identified in an evaluation of 2366 small molecules, the assay is envisioned to be adaptable to larger compound libraries. The assay is shown to be compatible with small-molecule DNA cleaving agents, such as bleomycin, neocarzinostatin chromophore, and lomaiviticin A, in place of IR.
Assuntos
Glicosídeos Cardíacos/farmacologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Imunofluorescência/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
The treatment of primary central nervous system tumors is challenging due to the blood-brain barrier and complex mutational profiles, which is associated with low survival rates. However, recent studies have identified common mutations in gliomas [isocitrate dehydrogenase (IDH)-wild-type and mutant, WHO grades II-IV; with grade IV tumors referred to as glioblastomas (GBM)]. These mutations drive epigenetic changes, leading to promoter methylation at the nicotinic acid phosphoribosyl transferase (NAPRT) gene locus, which encodes an enzyme involved in generating NAD+. Importantly, NAPRT silencing introduces a therapeutic vulnerability to inhibitors targeting another NAD+ biogenesis enzyme, nicotinamide phosphoribosyl transferase (NAMPT), rationalizing a treatment for these malignancies. Multiple systemically administered NAMPT inhibitors (NAMPTi) have been developed and tested in clinical trials, but dose-limiting toxicities-including bone marrow suppression and retinal toxicity-have limited their efficacy. Here, we report a novel approach for the treatment of NAPRT-silenced GBMs using nanoparticle (NP)-encapsulated NAMPTis administered by convection-enhanced delivery (CED). We demonstrate that GMX1778 (a NAMPTi) can be formulated in degradable polymer NPs with retention of potency for NAMPT inhibition and anticancer activity in vitro, plus sustained drug release in vitro and in vivo. Direct injection of these drugs via CED into the brain is associated with reduced retinal toxicity compared with systemic administration. Finally, we show that CED of NP-encapsulated GMX1778 to NAPRT-silenced intracranial GBM xenografts in mice exhibit significant tumor growth delay and extends survival. These data support an approach to treat gliomas harboring defects in NAD+ metabolism using CED of NP-encapsulated NAMPTis to greatly improve the therapeutic index and treatment efficacy for this class of drugs.
Assuntos
Glioma , Nanopartículas , Nicotinamida Fosforribosiltransferase , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Humanos , Animais , Camundongos , Nanopartículas/química , Glioma/tratamento farmacológico , Glioma/patologia , Citocinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologiaRESUMO
Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by loss of function mutations in fumarate hydratase (FH) and results in an aggressive subtype of renal cell carcinoma with limited treatment options. Loss of FH leads to accumulation of fumarate, an oncometabolite that disrupts multiple cellular processes and drives tumor progression. High levels of fumarate inhibit alpha ketoglutarate-dependent dioxygenases, including the ten eleven translocation (TET) enzymes and can lead to global DNA hypermethylation. Here, we report patterns of hypermethylation in FH-mutant cell lines and tumor samples are associated with silencing of nicotinate phosphoribosyl transferase (NAPRT), a rate-limiting enzyme in the Preiss-Handler pathway of NAD+ biosynthesis in a subset of HLRCC cases. NAPRT is hypermethylated at a CpG island in the promoter in cell line models and patient samples, resulting in loss of NAPRT expression. We find that FH-deficient RCC models with loss of NAPRT expression, as well as other oncometabolite-producing cancer models that silence NAPRT, are extremely sensitive to nicotinamide phosphoribosyl transferase inhibitors (NAMPTis). NAPRT silencing was also associated with synergistic tumor cell killing with poly(ADP)-ribose polymerase inhibitors (PARPis) and NAMPTis, which was associated with effects on PAR-mediated DNA repair. Overall, our findings indicate that NAPRT-silencing can be targeted in oncometabolite-producing cancers and elucidates how oncometabolite associated hypermethylation can impact diverse cellular processes and leads to therapeutically relevant vulnerabilities in cancer cells. Implications: NAPRT is a novel biomarker for targeting NAD+ metabolism in FH-deficient HLRCCs with NAMPTis alone and targeting DNA repair processes with the combination of NAMPTis and PARPis.
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The methylation status of the O6-methylguanine methyltransferase (MGMT) gene promoter has been widely accepted as a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ). In the absence of promoter methylation, the MGMT enzyme removes O6-methylguanine (O6-meG) lesions. In the setting of MGMT-promoter methylation (MGMT-), the O6-meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Our group reported that loss of MGMT expression via MGMT promoter silencing modulates activation of ataxia telangiectasia and RAD3 related protein (ATR) in response to TMZ treatment, which is associated with synergistic tumor-cell killing. Whether or not MMR proteins are involved in ATR activation in MGMT-cells upon alkylation damage remains poorly understood. To investigate the function of MMR in ATR activation, we created isogenic cell lines with knockdowns of the individual human MMR proteins MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutS homolog 3 (MSH3), MutL homolog 1 (MLH1), and PMS1 homolog 2 (PMS2). Here, we demonstrate that MSH2, MSH6, MLH1 and PMS2, specifically, are involved in the activation of the ATR axis after TMZ exposure, whereas MSH3 is likely not. This study elucidates a potential mechanistic understanding of how the MMR system is involved in ATR activation by TMZ in glioblastoma cells, which is important for targeting MMR-mutated cancers.
Assuntos
Glioblastoma , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Metiltransferases/metabolismo , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismoRESUMO
Approximately half of glioblastoma and more than two-thirds of grade II and III glioma tumors lack the DNA repair protein O6-methylguanine methyl transferase (MGMT). MGMT-deficient tumors respond initially to the DNA methylation agent temozolomide (TMZ) but frequently acquire resistance through loss of the mismatch repair (MMR) pathway. We report the development of agents that overcome this resistance mechanism by inducing MMR-independent cell killing selectively in MGMT-silenced tumors. These agents deposit a dynamic DNA lesion that can be reversed by MGMT but slowly evolves into an interstrand cross-link in MGMT-deficient settings, resulting in MMR-independent cell death with low toxicity in vitro and in vivo. This discovery may lead to new treatments for gliomas and may represent a new paradigm for designing chemotherapeutics that exploit specific DNA repair defects.
Assuntos
Antineoplásicos Alquilantes , Neoplasias Encefálicas , Metilases de Modificação do DNA , Enzimas Reparadoras do DNA , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Proteínas Supressoras de Tumor , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/genéticaRESUMO
Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody-drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.
Assuntos
DNA Topoisomerases Tipo I , Neoplasias , Inibidores da Topoisomerase I , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/análogos & derivados , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Topoisomerase I/farmacologiaRESUMO
Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) is mutated frequently in gliomas and represents a potential target for cancer therapies. ATRX is known to function as a histone chaperone that helps incorporate histone variant, H3.3, into the genome. Studies have implicated ATRX in key DNA damage response (DDR) pathways but a distinct role in DNA repair has yet to be fully elucidated. To further investigate the function of ATRX in the DDR, we created isogenic wild-type (WT) and ATRX knockout (KO) model cell lines using CRISPR-based gene targeting. These studies revealed that loss of ATRX confers sensitivity to poly(ADP)-ribose polymerase (PARP) inhibitors, which was linked to an increase in replication stress, as detected by increased activation of the ataxia telangiectasia and Rad3-related (ATR) signaling axis. ATRX mutations frequently co-occur with mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2), and the latter mutations also induce HR defects and PARP inhibitor sensitivity. We found that the magnitude of PARP inhibitor sensitivity was equal in the context of each mutation alone, although no further sensitization was observed in combination, suggesting an epistatic interaction. Finally, we observed enhanced synergistic tumor cell killing in ATRX KO cells with ATR and PARP inhibition, which is commonly seen in HR-defective cells. Taken together, these data reveal that ATRX may be used as a molecular marker for DDR defects and PARP inhibitor sensitivity, independent of IDH1/2 mutations. These data highlight the important role of common glioma-associated mutations in the regulation of DDR, and novel avenues for molecularly guided therapeutic intervention.
RESUMO
Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as 'BRCAness', which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.
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Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.
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[This corrects the article DOI: 10.1093/narcan/zcab021.].
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Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.
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O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide, providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches that could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. Temozolomide, unlike other alkylators, activated the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. Temozolomide induced growth delay, DNA double-strand breaks, and G2-M cell-cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with temozolomide increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and temozolomide. As ATR inhibitors are currently being tested in clinical trials, and temozolomide is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers. SIGNIFICANCE: Monofunctional alkylating agents sensitize MGMT-deficient tumor cells to ATR inhibitors.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Isoxazóis/farmacologia , Pirazinas/farmacologia , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Alquilantes/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Feminino , Humanos , Isoxazóis/administração & dosagem , Camundongos Nus , Pirazinas/administração & dosagem , Temozolomida/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pediatric high-grade gliomas are among the deadliest of childhood cancers due to limited knowledge of early driving events in their gliomagenesis and the lack of effective therapies available. In this study, we investigate the oncogenic role of PPM1D, a protein phosphatase often found truncated in pediatric gliomas such as DIPG, and uncover a synthetic lethal interaction between PPM1D mutations and nicotinamide phosphoribosyltransferase (NAMPT) inhibition. Specifically, we show that mutant PPM1D drives hypermethylation of CpG islands throughout the genome and promotes epigenetic silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key gene involved in NAD biosynthesis. Notably, PPM1D mutant cells are shown to be sensitive to NAMPT inhibitors in vitro and in vivo, within both engineered isogenic astrocytes and primary patient-derived model systems, suggesting the possible application of NAMPT inhibitors for the treatment of pediatric gliomas. Overall, our results reveal a promising approach for the targeting of PPM1D mutant tumors, and define a critical link between oncogenic driver mutations and NAD metabolism, which can be exploited for tumor-specific cell killing.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Nicotinamida Fosforribosiltransferase/genética , Proteína Fosfatase 2C/genética , Animais , Antineoplásicos/uso terapêutico , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Criança , Citocinas/antagonistas & inibidores , Metilação de DNA , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/patologia , Repressão Epigenética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , Ponte/citologia , Ponte/patologia , Cultura Primária de Células , Proteína Fosfatase 2C/metabolismo , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alzheimer's Disease (AD) is caused by the deposition of insoluble and toxic amyloid peptides (Abeta) in the brain leading to memory loss and other associated neurodegenerative symptoms. To date there is limited treatment options and strategies for treating AD. Studies have shown that clearance of the amyloid plaques from the brain and thus from the blood could be effective in stopping and or delaying the progression of the disease. Small peptides derived from the Abeta-42 sequence, in particular KLVFF, have shown to be effective binders of Abeta peptides and thus could be useful in delaying progression of the disease. We have taken advantage of this property by generating the retro-inverso (RI) version of this peptide, ffvlk, in different formats. We are presenting a new detox gel system using poly ethylene glycol (PEG), polymerized and cross linked with the RI peptides. We hypothesize that detox gel incorporating RI peptides will act like a 'sink' to capture the Abeta peptides from the surrounding environment. We tested these detox gels for their ability to capture biotinylated Abeta-42 peptides in vitro. The results showed that the detox gels bound Abeta-42 peptides effectively and irreversibly. Gels incorporating the tetramer RI peptide exhibited maximum binding capacity. The detox gel could be a potential candidate for treatment strategies to deplete the brain of toxic amyloid peptides.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/isolamento & purificação , Hidrogéis/administração & dosagem , Desintoxicação por Sorção/métodos , Doença de Alzheimer/metabolismo , Aminoácidos/análise , Peptídeos beta-Amiloides/metabolismo , Biotinilação , Implantes de Medicamento , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrogéis/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Relação Estrutura-AtividadeRESUMO
The hereditary cancer syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and succinate dehydrogenase-related hereditary paraganglioma and pheochromocytoma (SDH PGL/PCC) are linked to germline loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase and succinate dehydrogenase, thus leading to elevated levels of fumarate and succinate, respectively1-3. Here, we report that fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway required for the resolution of DNA double-strand breaks (DSBs) and for the maintenance of genomic integrity, thus rendering tumor cells vulnerable to synthetic-lethal targeting with poly(ADP)-ribose polymerase (PARP) inhibitors. These results identify HLRCC and SDH PGL/PCC as familial DNA-repair deficiency syndromes, providing a mechanistic basis to explain their cancer predisposition and suggesting a potentially therapeutic approach for advanced HLRCC and SDH PGL/PCC, both of which are incurable when metastatic.
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Ciclo do Ácido Cítrico/genética , Síndromes Neoplásicas Hereditárias/genética , Reparo de DNA por Recombinação , Neoplasias das Glândulas Suprarrenais/genética , Linhagem Celular , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Fumaratos/farmacologia , Mutação em Linhagem Germinativa , Células HEK293 , Humanos , Leiomiomatose/genética , Feocromocitoma/genética , Neoplasias Cutâneas/genética , Ácido Succínico/farmacologia , Neoplasias Uterinas/genéticaRESUMO
Intracranial delivery of therapeutic agents is limited by penetration beyond the blood-brain barrier (BBB) and rapid metabolism of the drugs that are delivered. Convection-enhanced delivery (CED) of drug-loaded nanoparticles (NPs) provides for local administration, control of distribution, and sustained drug release. While some investigators have shown that repeated CED procedures are possible, longer periods of sustained release could eliminate the need for repeated infusions, which would enhance safety and translatability of the approach. Here, we demonstrate that nanoparticles formed from poly(ethylene glycol)-poly(ω-pentadecalactone-co-p-dioxanone) block copolymers [PEG-poly(PDL-co-DO)] are highly efficient nanocarriers that provide long-term release: small nanoparticles (less than 100â¯nm in diameter) continuously released a radiosensitizer (VE822) over a period of several weeks in vitro, provided widespread intracranial drug distribution during CED, and yielded significant drug retention within the brain for over 1 week. One advantage of PEG-poly(PDL-co-DO) nanoparticles is that hydrophobicity can be tuned by adjusting the ratio of hydrophobic PDL to hydrophilic DO monomers, thus making it possible to achieve a wide range of drug release rates and drug distribution profiles. When administered by CED to rats with intracranial RG2 tumors, and combined with a 5-day course of fractionated radiation therapy, VE822-loaded PEG-poly(PDL-co-DO) NPs significantly prolonged survival when compared to free VE822. Thus, PEG-poly(PDL-co-DO) NPs represent a new type of versatile nanocarrier system with potential for sustained intracranial delivery of therapeutic agents to treat brain tumors.
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Materiais Biocompatíveis/química , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Poliésteres/química , Polietilenoglicóis/química , Animais , Neoplasias Encefálicas/patologia , Convecção , Liberação Controlada de Fármacos , Hidrodinâmica , Isoxazóis/farmacologia , Masculino , Nanopartículas/ultraestrutura , Poliésteres/síntese química , Polietilenoglicóis/síntese química , Pirazinas/farmacologia , Radiossensibilizantes/farmacologia , Ratos Endogâmicos F344 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
High-grade gliomas, such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), are characterized by an aggressive phenotype with nearly universal local disease progression despite multimodal treatment, which typically includes chemotherapy, radiotherapy, and possibly surgery. Radiosensitizers that have improved the effects of radiotherapy for extracranial tumors have been ineffective for the treatment of GBM and DIPG, in part due to poor blood-brain barrier penetration and rapid intracranial clearance of small molecules. Here, we demonstrate that nanoparticles can provide sustained drug release and minimal toxicity. When administered locally, these nanoparticles conferred radiosensitization in vitro and improved survival in rats with intracranial gliomas when delivered concurrently with a 5-day course of fractionated radiotherapy. Compared with previous work using locally delivered radiosensitizers and cranial radiation, our approach, based on the rational selection of agents and a clinically relevant radiation dosing schedule, produces the strongest synergistic effects between chemo- and radiotherapy approaches to the treatment of high-grade gliomas. Mol Cancer Ther; 16(8); 1456-69. ©2017 AACR.
Assuntos
Neoplasias do Tronco Encefálico/tratamento farmacológico , Reparo do DNA , Glioma/tratamento farmacológico , Radiossensibilizantes/uso terapêutico , Animais , Neoplasias do Tronco Encefálico/patologia , Linhagem Celular Tumoral , Convecção , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Glioma/patologia , Humanos , Masculino , Nanopartículas/química , Nanopartículas/ultraestrutura , Poliésteres/química , Polietilenoglicóis/química , Radiossensibilizantes/farmacologia , Ratos Endogâmicos F344 , Distribuição Tecidual/efeitos dos fármacosRESUMO
DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.
Assuntos
Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Proteína 1 com Domínio SAM e Domínio HD/genética , Quebras de DNA de Cadeia Dupla , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , TransfecçãoRESUMO
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 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.