Dataset Information

MTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

ABSTRACT: The mammalian target of rapamycin (mTOR) is a kinase that functions in two distinct complexes, mTORC1 and mTORC2. In peripheral B cells, complete deletion of mTOR suppresses germinal center B-cell responses, including class switching and somatic hypermutation. The allosteric mTORC1 inhibitor rapamycin blocks proliferation and differentiation, but lower doses can promote protective IgM responses. To elucidate the complexity of mTOR signaling in B cells further, we used ATP-competitive mTOR kinase inhibitors (TOR-KIs), which inhibit both mTORC1 and mTORC2. Although TOR-KIs are in clinical development for cancer, their effects on mature lymphocytes are largely unknown. We show that high concentrations of TOR-KIs suppress B-cell proliferation and differentiation, yet lower concentrations that preserve proliferation increase the fraction of B cells undergoing class switching in vitro. Transient treatment of mice with the TOR-KI compound AZD8055 increased titers of class-switched high-affinity antibodies to a hapten-protein conjugate. Mechanistic investigation identified opposing roles for mTORC1 and mTORC2 in B-cell differentiation and showed that TOR-KIs enhance class switching in a manner dependent on forkhead box, subgroup O (FoxO) transcription factors. These observations emphasize the distinct actions of TOR-KIs compared with rapamycin and suggest that TOR-KIs might be useful to enhance production of class-switched antibodies following vaccination.

SUBMITTER: Limon JJ

PROVIDER: S-EPMC4250172 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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mTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

Limon Jose J JJ So Lomon L Jellbauer Stefan S Chiu Honyin H Corado Juana J Sykes Stephen M SM Raffatellu Manuela M Fruman David A DA

Proceedings of the National Academy of Sciences of the United States of America 20141110 47

The mammalian target of rapamycin (mTOR) is a kinase that functions in two distinct complexes, mTORC1 and mTORC2. In peripheral B cells, complete deletion of mTOR suppresses germinal center B-cell responses, including class switching and somatic hypermutation. The allosteric mTORC1 inhibitor rapamycin blocks proliferation and differentiation, but lower doses can promote protective IgM responses. To elucidate the complexity of mTOR signaling in B cells further, we used ATP-competitive mTOR kinase ...[more]

PMID: 25385646

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Project description:The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)-->Akt-->mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.

Project description:Despite microsurgical repair, recovery of function following peripheral nerve injury is slow and often incomplete. Outcomes could be improved by an increased understanding of the molecular biology of regeneration and by translation of experimental bioengineering strategies. Topographical cues have been shown to be powerful regulators of the rate and directionality of neurite regeneration, and in this study we investigated the downstream molecular effects of linear micropatterned structures in an organotypic explant model. Linear topographical cues enhanced neurite outgrowth and our results demonstrated that the mTOR pathway is important in regulating these responses. mTOR gene expression peaked between 48 and 72h, coincident with the onset of rapid neurite outgrowth and glial migration, and correlated with neurite length at 48h. mTOR protein was located to glia and in a punctate distribution along neurites. mTOR levels peaked at 72h and were significantly increased by patterned topography (p<0.05). Furthermore, the topographical cues could override pharmacological inhibition. Downstream phosphorylation assays and inhibition of mTORC1 using rapamycin highlighted mTORC2 as an important mediator, and more specific therapeutic target. Quantitative immunohistochemistry confirmed the presence of the mTORC2 component rictor at the regenerating front where it co-localised with F-actin and vinculin. Collectively, these results provide a deeper understanding of the mechanism of action of topography on neural regeneration, and support the incorporation of topographical patterning in combination with pharmacological mTORC2 potentiation within biomaterial constructs used to repair peripheral nerves.Statement of significancePeripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.

Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.

Project description:Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) C? constant region exons (CHs) with one of six CHs lying 100-200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region1. Cytokines and activators induce activation-induced cytidine deaminase (AID)2 and I-promoter transcription, with 3' IgH regulatory region (3' IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3' IgHRR interactions3-8. Transcription through donor S? and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs9-11. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an S?-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3' IgHRR enhancers with the 200-kb upstream S? to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with S? for deletional CSR. During constitutive S? CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with S? in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

Dataset Information

MTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

Publications

mTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

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