Targeting LMO2 with a Peptide Aptamer Establishes a Necessary Function in Overt T-Cell Neoplasia

Appert, Alex; Nam, Chang-Hoon; Lobato, Natividad; Priego, Eva-Marı́a; Miguel, Ricardo Núñez; Blundell, Tom L.; Drynan, Lesley; Sewell, Helen; Tanaka, Tomoyuki; Rabbitts, Terence H.

doi:10.1158/0008-5472.can-08-4774

Cited by 36 publications

(48 citation statements)

References 45 publications

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“…It has been previously shown possible to inhibit ETS factors in whole animal models without any harm coming to normal tissue (Huang et al, 2006). As high LMO2 removal is sufficient to cause death of leukaemic cells even after acquisition of secondary genetic abnormalities (Appert et al, 2009), identification of the molecular mechanisms involved in aberrant ectopic expression of LMO2 may open up new targeted therapeutic options directed towards the impediment of upstream regulators to block the maintenance of the leukaemia phenotype.…”

Section: Discussionmentioning

confidence: 99%

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients

et al. 2010

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The T-cell oncogene Lim-only 2 (LMO2) critically influences both normal and malignant haematopoiesis. LMO2 is not normally expressed in T cells, yet ectopic expression is seen in the majority of T-acute lymphoblastic leukaemia (T-ALL) patients with specific translocations involving LMO2 in only a subset of these patients. Ectopic lmo2 expression in thymocytes of transgenic mice causes T-ALL, and retroviral vector integration into the LMO2 locus was implicated in the development of clonal T-cell disease in patients undergoing gene therapy. Using array-based chromatin immunoprecipitation, we now demonstrate that in contrast to B-acute lymphoblastic leukaemia, human T-ALL samples largely use promoter elements with little influence from distal enhancers. Active LMO2 promoter elements in T-ALL included a previously unrecognized third promoter, which we demonstrate to be active in cell lines, primary T-ALL patients and transgenic mice. The ETS factors ERG and FLI1 previously implicated in lmo2-dependent mouse models of T-ALL bind to the novel LMO2 promoter in human T-ALL samples, while in return LMO2 binds to blood stem/progenitor enhancers in the FLI1 and ERG gene loci. Moreover, LMO2, ERG and FLI1 all regulate the þ 1 enhancer of HHEX/PRH, which was recently implicated as a key mediator of early progenitor expansion in LMO2-driven T-ALL. Our data therefore suggest that a self-sustaining triad of LMO2/ERG/FLI1 stabilizes the expression of important mediators of the leukaemic phenotype such as HHEX/PRH.

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Section: Discussionmentioning

confidence: 99%

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients

et al. 2010

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“…Oncogenic addiction infers that secondary mutations are unlikely to replace the function of the initiating oncogenic event. Accordingly, it has been shown that Lmo2 function is essential for the maintenance of Lmo2-induced T-ALL in mice (36).…”

Section: Clinical-translational Advancesmentioning

confidence: 99%

The Molecular Basis of Lmo2-Induced T-Cell Acute Lymphoblastic Leukemia

Curtis

McCormack

2010

Clinical Cancer Research

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T-cell acute lymphoblastic leukemia (T-ALL) is commonly caused by the overexpression of oncogenic transcription factors in developing T cells. In a mouse model of one such oncogene, LMO2, the cellular effect is to induce self-renewal of committed T cells in the thymus, which persist long-term while acquiring additional mutations and eventually giving rise to leukemia. These precancerous stem cells (pre-CSC) are intrinsically resistant to radiotherapy, implying that they may be refractory to conventional cancer therapies. However, they depend on an aberrantly expressed stem cell-like self-renewal program for their maintenance, in addition to a specialized thymic microenvironmental niche. Here, we discuss potential approaches for targeting pre-CSCs in T-ALL by using therapies directed at oncogenic transcription factors themselves, downstream self-renewal pathways, and the supportive cell niche. Clin Cancer Res; 16(23); 5618-23. Ó2010 AACR.

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“…2), and an analogous phenomenon has been reported for anti-LMO2 VL segments, which may be iDAbs that bind to zinc-binding regions of LMO2. A related effect was found with an anti-LMO2 peptide aptamer described previously that seems to interact with the zinc in LMO2 LIM finger 4 (30). IAC 3 -derived Single Domains Facilitate Recombinant Protein Production for Structural Analysis-X-ray crystallography is a key technique in facilitating the determination of the structures of many important proteins and complexes to help elucidate biological mechanisms.…”

Section: Advantages Of Iacmentioning

confidence: 80%

“…VH#576 Hinders LMO2-dependent T-cell Neoplasia-LMO2 was originally discovered because it is activated in T-cell acute leukemia by chromosomal translocations (28,29) and, using macrodrugs such as scFv and peptide aptamers, we have shown that it is required for tumor growth in transgenic mouse models of Lmo2-dependent neoplasia (11,30). We sought to further validate the anti-LMO2 VH#576 utilizing FIGURE 3.…”

Section: Iac 3 -Selected Single Domains Function In Mammalian Cells-tmentioning

confidence: 99%

Single Domain Intracellular Antibodies from Diverse Libraries

Tanaka

Sewell

Waters

et al. 2011

Journal of Biological Chemistry

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Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC 3 ) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Cocrystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC 3 offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.Antibodies and derivative fragments are molecules that can capture a huge variety of antigens with high specificity and affinity and are indispensable tools and reagents in bioscience and in medicine (1). The utilities of intracellular application of antibody fragments is that, unlike gene knock-out and interfering RNAs or antisense, these molecules interfere with cellular functions directly by binding to target proteins and can therefore target specific properties of proteins such as particular signal transduction via blockading protein-protein interaction (2, 3). This not only provides valuable and robust tools in biological analysis such as functional genomics and proteomics but also serve as drug surrogates to establish the target validity for drug development by ascertaining the biological effects from targeting the protein. To make these options possible on a broad basis, robust screening methods with high quality libraries are needed to allow selection of target-specific intracellular antibodies.Antibody binding sites comprise heavy chain variable domain (VH) 4 and light chain variable domains (VL), each with three complementarity determining regions (CDRs), directly contacting antigen and concerned in binding affinity and specificity. Single domain antibody fragments (DAbs), comprising either VH or VL, encompass a minimal antigen recognition can also be used for intracellular applications, so-called intracellular domain antibodies (iDAbs) (4). Structural analysis of V regions based on a consensus framework sequence have shown that essentially no diff...

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Targeting LMO2 with a Peptide Aptamer Establishes a Necessary Function in Overt T-Cell Neoplasia

Cited by 36 publications

References 45 publications

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients

The Molecular Basis of Lmo2-Induced T-Cell Acute Lymphoblastic Leukemia

Single Domain Intracellular Antibodies from Diverse Libraries

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