ABSTRACT
Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.
Subject(s)
Genomic Instability , Rad51 Recombinase , Animals , Humans , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Genomic Instability/genetics , Homologous Recombination/genetics , DNA Breaks, Double-Stranded , RNA , DNA Repair/genetics , Mammals/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolismABSTRACT
DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Binding Proteins/metabolism , Mad2 Proteins/metabolism , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , G2 Phase/genetics , HEK293 Cells , Humans , Mad2 Proteins/genetics , S Phase/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eµ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.
Subject(s)
CRISPR-Associated Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Techniques , Animals , CRISPR-Associated Proteins/metabolism , Female , Gene Targeting , Genes, p53/genetics , Genome/genetics , INDEL Mutation/genetics , Kaplan-Meier Estimate , Lentivirus/genetics , Lymphoma/genetics , Lymphoma/mortality , Lymphoma/therapy , Mice , Mice, Inbred C57BL , Mutation , Reproducibility of ResultsABSTRACT
Aims: The biogenesis of high-density lipoprotein (HDL) particles by cholesterol-laden foam cells in atherosclerotic lesions is crucial for the removal of excess cholesterol from the lesions. Impairment in the HDL biogenic process contributes to the progression of atherosclerosis. The aim of this study is to identify novel cellular factors regulating HDL biogenesis. Methods and results: HDL biogenesis is a process of apolipoprotein (apo)-mediated solubilization of specific plasma membrane (PM) microdomains generated in cholesterol-accumulated cells. We established a new method to isolate PM microdomains interacting with the major HDL protein constituent, apoA-I. Lipidomic and proteomic analyses of an isolated PM microdomain revealed that apoA-I binds to cholesterol-rich and desmocollin 1 (DSC1)-containing microdomains. In this novel apoA-I binding microdomain, DSC1 binds and prevents apoA-I from interacting with another PM microdomain created by adenosine triphosphate-binding cassette transporter A1 (ABCA1) for the formation of HDL. Inhibition of apoA-I-DSC1 binding by silencing DSC1 expression or using DSC1 blocking antibodies increases apoA-I accessibility to ABCA1-created microdomains and thus enhances HDL biogenesis. Importantly, DSC1 is abundantly expressed in macrophages and human atherosclerotic lesions, suggesting that DSC1 may contribute to cholesterol accumulation in atherosclerotic lesions by sequestering apoA-I and impairing HDL biogenesis. Conclusions: The binding of apoA-I to two functionally opposing PM microdomains, ABCA1 and DSC1 domains, suggests that HDL biogenesis and PM cholesterol levels may be regulated by the relative abundance of the two domains and that novel HDL biogenic therapies may be developed by targeting DSC1.
Subject(s)
Atherosclerosis/metabolism , Desmocollins/metabolism , Lipoproteins, HDL/biosynthesis , Apolipoprotein A-I/metabolism , Binding Sites , Gene Expression Regulation , HEK293 Cells , Humans , Lipoproteins, HDL/metabolism , Protein BindingABSTRACT
DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest. This is accompanied by morphological defects, elevation of senescence-associated ß-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression.
Subject(s)
Cell Cycle Checkpoints/physiology , Cellular Senescence/physiology , DEAD-box RNA Helicases/metabolism , DNA Replication/physiology , Fibroblasts/metabolism , Neoplasm Proteins/metabolism , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , DEAD-box RNA Helicases/genetics , Diploidy , Fibroblasts/cytology , HEK293 Cells , Humans , Neoplasm Proteins/genetics , Tumor Suppressor Protein p53/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737-shDhx9 synthetic lethal relationship.
Subject(s)
Biphenyl Compounds/pharmacology , DEAD-box RNA Helicases/genetics , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Lymphoma/genetics , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , Sulfonamides/pharmacology , Animals , Cell Cycle , Cyclin-Dependent Kinase Inhibitor p16/physiology , DEAD-box RNA Helicases/antagonists & inhibitors , DEAD-box RNA Helicases/metabolism , Genes, Modifier , Humans , Lymphoma/pathology , Mice , Myeloid Cell Leukemia Sequence 1 Protein , Piperazines/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.
Subject(s)
DNA Repair , Rad51 Recombinase , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Proteins/genetics , DNA/genetics , MitosisABSTRACT
Mcl-1, a pro-survival member of the Bcl-2 family located at the mitochondrial outer membrane, is subject to constitutive ubiquitylation by the Bcl-2 homology 3-only E3 ligase, Mule/Lasu1, resulting in rapid steady-state degradation via the proteasome. Insertion of newly synthesized Mcl-1 into the mitochondrial outer membrane is dependent on its C-terminal transmembrane segment, but once inserted, the N terminus of a portion of the Mcl-1 molecules can be subject to proteolytic processing. Remarkably, this processing requires an intact electrochemical potential across the inner membrane. Three lines of evidence directed at the endogenous protein, however, indicate that the resulting Mcl-1ΔN isoform resides in the outer membrane: (i) full-length Mcl-1 and Mcl-1ΔN resist extraction by alkali but are accessible to exogenous protease; (ii) almost the entire populations of Mcl-1 and Mcl-1ΔN are accessible to the membrane-impermeant Cys-reactive agent 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid; and (iii) Mcl-1 and Mcl-1ΔN exhibit equivalent chemical cross-linking to Bak in intact mitochondria, an Mcl-1 binding partner located in the outer membrane. In addition to the Mule Bcl-2 homology 3 domain, we show that interaction between Mcl-1 and Mule also requires the extreme N terminus of Mcl-1, which is lacking in Mcl-1ΔN. Thus, Mcl-1ΔN does not interact with Mule, exhibits reduced steady-state ubiquitylation, evades the hyper-rapid steady-state degradation that is observed for full-length Mcl-1 in response to treatments that limit global protein synthesis, and confers resistance to UV stress-induced cell death.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Proteolysis , Proto-Oncogene Proteins c-bcl-2/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Binding Sites , Cell Death/physiology , Cell Death/radiation effects , HeLa Cells , Humans , Mice , Mice, Knockout , Mitochondria/genetics , Myeloid Cell Leukemia Sequence 1 Protein , NIH 3T3 Cells , Protein Biosynthesis/physiology , Protein Biosynthesis/radiation effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases/genetics , Ubiquitination/physiology , Ubiquitination/radiation effects , Ultraviolet RaysABSTRACT
Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is a frequent occurrence in human cancers and a major promoter of chemotherapeutic resistance. Inhibition of one downstream target in this pathway, mTORC1, has shown potential to improve chemosensitivity. However, the mechanisms and genetic modifications that confer sensitivity to mTORC1 inhibitors remain unclear. Here, we demonstrate that loss of TSC2 in the E mu-myc murine lymphoma model leads to mTORC1 activation and accelerated oncogenesis caused by a defective apoptotic program despite compromised AKT phosphorylation. Tumors from Tsc2(+/-)E mu-Myc mice underwent rapid apoptosis upon blockade of mTORC1 by rapamycin. We identified myeloid cell leukemia sequence 1 (Mcl-1), a bcl-2 like family member, as a translationally regulated genetic determinant of mTORC1-dependent survival. Our results indicate that the extent by which rapamycin can modulate expression of Mcl-1 is an important feature of the rapamycin response.
Subject(s)
Gene Expression Regulation, Neoplastic/physiology , Lymphoma/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/physiology , Sirolimus/metabolism , Transcription Factors/metabolism , Animals , Gene Expression Regulation, Neoplastic/drug effects , Immunoblotting , Immunoprecipitation , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes , Myeloid Cell Leukemia Sequence 1 Protein , Proteins , Reverse Transcriptase Polymerase Chain Reaction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Transcription Factors/antagonists & inhibitors , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Evading apoptosis is considered to be a hallmark of cancer, because mutations in apoptotic regulators invariably accompany tumorigenesis. Many chemotherapeutic agents induce apoptosis, and so disruption of apoptosis during tumour evolution can promote drug resistance. For example, Akt is an apoptotic regulator that is activated in many cancers and may promote drug resistance in vitro. Nevertheless, how Akt disables apoptosis and its contribution to clinical drug resistance are unclear. Using a murine lymphoma model, we show that Akt promotes tumorigenesis and drug resistance by disrupting apoptosis, and that disruption of Akt signalling using the mTOR inhibitor rapamycin reverses chemoresistance in lymphomas expressing Akt, but not in those with other apoptotic defects. eIF4E, a translational regulator that acts downstream of Akt and mTOR, recapitulates Akt's action in tumorigenesis and drug resistance, but is unable to confer sensitivity to rapamycin and chemotherapy. These results establish Akt signalling through mTOR and eIF4E as an important mechanism of oncogenesis and drug resistance in vivo, and reveal how targeting apoptotic programmes can restore drug sensitivity in a genotype-dependent manner.
Subject(s)
Apoptosis , Drug Resistance, Neoplasm , Eukaryotic Initiation Factor-4E/metabolism , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/pathology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Signal Transduction , Animals , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Apoptosis/drug effects , Cell Division/drug effects , Cell Survival/drug effects , Disease Progression , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4G/metabolism , Female , Lymphoma, B-Cell/metabolism , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Protein Kinase Inhibitors , Protein Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The phosphatidylinositol-3-OH kinase [PI(3)K] pathway is frequently activated in human cancers and represents a rational target for therapeutic intervention. We have previously shown that enforced expression of Akt, which is a downstream effector of PI(3)K, could promote tumorigenesis and drug resistance in the Emu-myc mouse lymphoma model, and that these tumors were particularly sensitive to inhibition of mammalian target of rapamycin (mTOR) with rapamycin when combined with conventional chemotherapy. We now show that reduced dosage of PTEN, a negative regulator of PI(3)K signaling, is sufficient to activate Akt, but has only a modest effect on lymphomagenesis in the same model. Nonetheless, loss of even one PTEN allele resulted in lymphomas that were resistant to conventional chemotherapy yet sensitive to rapamycin/chemotherapy combinations. These effects could be recapitulated by using RNA interference to suppress PTEN expression in lymphomas, which were previously established in the absence of PI(3)K lesions. Finally, the introduction of lesions that act downstream of mTOR (eIF4E) or disable apoptosis (Bcl-2 and loss of p53) into PTEN+/- lymphomas promoted resistance to rapamycin/chemotherapy combinations. Thus, whether activation of the PI(3)K pathway confers sensitivity or resistance to therapy depends on the therapy used as well as secondary genetic events. Understanding these genotype-response relationships in human tumors will be important for the effective use of rapamycin or other compounds targeting the PI(3)K pathway in the clinic.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Lymphoma/drug therapy , Animals , Drug Resistance, Neoplasm , Enzyme Activation , Female , Genes, myc , Heterozygote , Lymphoma/genetics , Lymphoma/metabolism , Male , Mice , Mice, Inbred C57BL , PTEN Phosphohydrolase/biosynthesis , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Sirolimus/administration & dosageABSTRACT
The use of small molecule inhibitors in the study of cellular processes is a powerful approach to understanding gene function. During the course of a high throughput screen for novel inhibitors of eukaryotic translation, we identified a number of nucleic acid binding ligands that showed activity in our assay. When tested on a panel of mRNA transcripts displaying different modes of translation initiation, these ligands showed a range of biological activities--with some inhibiting both cap-dependent and internal initiation and others preferentially blocking internal initiation. We used this information to identify a novel threading intercalator that inhibits Hepatitis C virus internal initiation.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Intercalating Agents/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Acridines/chemistry , Acriflavine/chemistry , Acriflavine/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Humans , Ligands , Peptidyl Transferases/antagonists & inhibitors , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/metabolism , Ribosomes/drug effects , Ribosomes/metabolism , Structure-Activity Relationship , Viral Proteins/biosynthesisABSTRACT
In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.
Subject(s)
CRISPR-Cas Systems , DNA Cleavage , Nucleotide Motifs , RNA Editing , RNA, Guide, Kinetoplastida/chemistry , Blotting, Northern , Blotting, Western , CRISPR-Associated Proteins , Electrophoretic Mobility Shift Assay , Genome , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , RNA/metabolismABSTRACT
BRCA2-associated pancreatic ductal adenocarcinoma (PDAC) may be sensitive to agents that target homology-directed DNA repair, such as DNA crosslinking agents (DCLs) and PARP inhibitors (PARPis). Here, we assessed the sensitivities of BRCA2-deficient (Capan-1) and BRCA2-proficient (MIA PaCa-2) PDAC cell lines to a panel of DCLs and PARPis. Compared to MIA PaCa-2, Capan-1 was significantly more sensitive to all tested DCLs and PARPis, with similar increased sensitivities to cisplatin and the PARPi BMN 673 compared to other DCLs and the PARPi veliparib. We provide further support for this observation by showing that shRNA-mediated BRCA2 knockdown in PANC-1, a BRCA2-proficient cell line, induces sensitization to cisplatin and BMN 673 but not to veliparib. These findings were validated in a PDAC murine xenograft model derived from a patient with bi-allelic BRCA2 mutations. We found 64% and 61% tumor growth inhibition of this xenograft with cisplatin and BMN 673 treatments, respectively. Cisplatin and BMN 673 treatments reduced cellular proliferation and induced apoptosis. Our findings support a personalized treatment approach for BRCA2-associated PDAC.
Subject(s)
Genes, BRCA2 , Pancreatic Neoplasms/drug therapy , Phthalazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors , Cell Line, Tumor , Cisplatin/therapeutic use , Gene Knockdown Techniques , Germ-Line Mutation , Humans , In Vitro Techniques , Male , Middle Aged , Pancreatic Neoplasms/geneticsABSTRACT
The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.
Subject(s)
CRISPR-Associated Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Genetic Testing/methods , Genetic Vectors/genetics , Genomics/methods , RNA, Guide, Kinetoplastida/genetics , Retroviridae/genetics , Animals , Base Sequence , CRISPR-Cas Systems , Genetic Vectors/administration & dosage , Humans , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Guide, Kinetoplastida/administration & dosage , Transduction, Genetic/methodsABSTRACT
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to (5')NGG(3') protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5' end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , DNA Cleavage , Animals , Base Sequence , Cells, Cultured , Chromatin Immunoprecipitation , Mice , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
BACKGROUND: The predominant mechanism by which human tumors maintain telomere length is via telomerase. In ~10% of tumor samples, however, telomere length is conserved, despite no detectable telomerase activity, in part through activation of the alternative lengthening of telomeres (ALT) pathway. METHODS: We studied the circular extra-chromosomal telomeric repeat (ECTR), an ALT hallmark, and telomerase activity in 24 chronic myeloid leukemia (CML) patients in chronic phase (CP). RESULTS: We identified the presence of ECTR in primary leukemia cells from some of these samples, which indicates the possible involvement of an ALT mechanism. Moreover, we found that some samples exhibited both circular ECTR and telomerase activities, suggesting that both mechanisms can contribute to the onset of CML. CONCLUSION: We propose that ALT or the combined activities of ALT and telomerase might be required for the early stages of leukemogenesis. These findings shed new light into the oncogenic pathways responsible for the maintenance of telomere length in leukemia, which will ultimately determine the effectiveness of anti-telomerase-based treatment protocols.
Subject(s)
Chromosome Aberrations , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Repetitive Sequences, Nucleic Acid/genetics , Telomerase/genetics , Telomere Homeostasis/genetics , Telomere/genetics , Humans , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Elimination of myeloid leukemia cell 1 (Mcl-1) is an early event in the onset of cell death following DNA damage and, in many settings, plays a critical role in dictating the success of chemotherapeutic agents. Following DNA damage, Mcl-1 is rapidly and efficiently targeted to the 26S proteasome through the action of E3 ubiquitin ligases. Tumors having acquired lesions that lead to stabilization of Mcl-1 are highly aggressive and have a poorer prognosis. Herein, we further characterize an additional mechanism of Mcl-1 proteolysis that is proteasome-independent but mitochondrial-dependent. A mitochondrial targeting signal located in the N terminus of Mcl-1 is essential for targeting Mcl-1 to this alternative degradative avenue. We demonstrate that the Akt/mTORC1 survival pathway protects Mcl-1 from mitochondrial-dependent proteolysis. Disrupting Mcl-1 inner mitochondrial targeting improves the pro-survival capacity of Mcl-1 both ex vivo and in vivo in the well-characterized mouse Eµ-Myc lymphoma model. Our data uncover an important relationship between the mitochondria and the Mcl-1 N terminus in dictating cell fate following DNA damage.
Subject(s)
DNA Damage , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Disease Models, Animal , Lymphoma/drug therapy , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mutagenesis , Myeloid Cell Leukemia Sequence 1 Protein , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
A defining feature of many cancers is deregulated translational control. Typically, this occurs at the level of recruitment of the 40S ribosomes to the 5'-cap of cellular messenger RNAs (mRNAs), the rate-limiting step of protein synthesis, which is controlled by the heterotrimeric eukaryotic initiation complex eIF4F. Thus, eIF4F in particular, and translation initiation in general, represent an exploitable vulnerability and unique opportunity for therapeutic intervention in many transformed cells. In this article, we discuss the development, mode of action and biological activity of a number of small-molecule inhibitors that interrupt PI3K/mTOR signaling control of eIF4F assembly, as well as compounds that more directly block eIF4F activity.
Subject(s)
Neoplasms/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Eukaryotic Initiation Factor-4F/metabolism , HumansABSTRACT
A challenge in cancer therapy is to selectively target activities that are essential for survival of malignant cells while sparing normal cells. Translational control represents a potential anti-neoplastic target because it is exerted by major signaling pathways that are often usurped in cancers. Herein we describe approaches being developed that target eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex that integrates multiple signaling inputs to the translation apparatus.