Function Of LMO2 Research Articles

Myelo-Erythroid Lineage Segregation Is Regulated By the GATA-2 Interactome

Hematopoiesis arises from hematopoietic stem cell differentiating into downstream progenitors through a multistep process of sequential lineage potential restriction leading to the final production of >10 hematopoietic cell type.The regulation of hematopoietic lineage development involves synergistic and antagonistic interactions between transcription factors (TF) with lineage-restricted expression patterns, both through protein-protein interaction and direct transcriptional activation.Eosinophils, basophils and mast cells are key myeloid cell types involved in allergic inflammation and have been proposed to arise from a granulocyte-macrophage progenitor (GMP), that also generates neutrophils and macrophages, with GATA-2 up-regulation as a critical determinant. However, recent work identified a distinct basophil/eosinophil/mast cells progenitor (BEMP) that arises from multi-potent erythroid-primed multipotent progenitors (EMPP) where GATA-2 is already expressed ( Drissen et al., 2016). Here, we show that the GATA-2 interactome, rather than GATA-2 expression, controls myeloid lineage choice in EMPPs. To investigate the molecular mechanism instructing lineage decision in EMPP, we performed gene-expression profiling (RNA-seq) comparing its downstream progeny, early restricted erythroid/megakaryocyte progenitor (preMegE) and BEMP. This analysis identified differential expressed genes, including antagonist gene-expression pattern between the LIM-only protein 2 ( Lmo2) and protein 4 ( Lmo4), both previously shown as GATA-2 interactor. While Lmo2 function in hematopoiesis have been exhaustively studied, the role of Lmo4 remains unknown. In colony forming unit (CFU)-assay, overexpression of Lmo4 in EMPP mediated by lentiviral delivery, resulted in an increased number of mast-colonies associated with a decreased of erythroid/megakaryocyte colonies, while the total colony number was unchanged. In addition, transplantation of hematopoietic lineage-negative cells transduced with Lmo4/empty(Ctrl) lentiviral particles, showed an increased frequency of basophils and eosinophils associated with a decrease of erythroid cells in Lmo4 overexpressing cells compared to Ctrl, in both bone marrow and spleen of recipient mice, 8 weeks after transplantation. Analysis of transduced Lineage-negative Kit + (LK) compartment of recipient mice, also revealed an increased frequency of BEMP associated with a decreased frequency of preMegE in Lmo4overexpressing cells compared to Ctrl. Taken together, these data suggested that Lmo4 overexpression instruct lineage decision of EMPP toward basophil/eosinophil/mast lineages.Over-expression followed by co-immunoprecipitation in HEK-293T cell line revealed that LMO4 compete with LMO2 binding to GATA-2, suggesting a mutually exclusive transcriptional complex. More importantly, lack of interaction between LMO4 and GATA-2 mediated by a point mutation in the zinc-finger 1 domain (ZnF1) of GATA-2 (GATA2-G320D), frequently identified in AML/AEL, resulted in an absence of mast lineage potential of EMPP compared to GATA2 +/+. Moreover, over-expression of Lmo4 in EMPP from GATA2 G320D/G320D mice compared to GATA2 +/+, failed to instruct myeloid fate in vitro. Overall, our data identified a specific molecular mechanism of hematopoietic cell fate decision driven by protein-protein interaction with GATA-2. Perturbation of GATA-2 interactome through over-expression of the cofactor Lmo4 or specific point mutation in GATA2 ZnF1 domain results in lineage fate switch of EMPP toward the erythroid/megakaryocyte or basophil/eosinophil/mast lineages.

Biochemical Feature of LMO2 Interactome and LMO2 Function Prospect.

BackgroundLMO2 belongs to the LIM-Only group of LIM domain protein superfamily. It is ubiquitously expressed in different types of tissues and locates either in the nucleus or in the cytoplasm depending on the tissue type. Till now the unique function of LMO2 was considered to be serving as a bridging or blocking molecule that mediates extensive protein-protein interactions. However, the exactly biological features of LMO2 interactome as well as LMO2 function spectrum remain largely unclear.Material/MethodsIn this study, yeast 2-hybrid assay was firstly performed using LMO2 as the bait and the characteristic of LMO2 protein interactome was analyzed according to the yeast 2-hybrid data and other relative biological information primarily using bioinformatic method.ResultsOur data indicated that LMO2 favored interacting with peptides containing β-sheet structure and having relatively unstable confirmation. Moreover, several LMO2 favored interacting domains were identified, including WD40 repeat, coiled-coil, Ankyrin repeat, Zinc finger, PDZ, and SH3, and functions of these domain-containing members were dramatically enriched in some types of cancers.ConclusionsOur results revealed a LMO2 favored protein-interaction pattern in both secondary structure and domain level, and concentrated LMO2 function in kinds of cytoplasmic metabolism pathways as well as multiple types of cancers.

Comprehensive Analysis of Immunophenotype and Transcriptome in Indian T-Acute Lymphoblastic Leukemia - a Prospective Study

T-cell acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors that result from the malignant transformation of T-cell progenitors. T-ALLs are classified immunologically into immature (pro- and pre-T), cortical and mature T-ALL. ETP-ALL is a recently described subtype of immature T-ALL. T-ALL is characterized by overexpression of oncogenic transcription factors that drive the malignancy: TAL1, TAL2, LYL1,LMO1, LMO2; TLX1, TLX3, NKX2.1, NKX2.2, NKX2.5, MEF2C and HOX genes. This study was conducted to understand the landscape of protein-coding genes using RNAseq in immature, cortical and mature T-ALL patients in India. This was a prospective study done on 134 T-ALL cases. The T-ALL patients were divided into 2 cohorts: discovery (n=35; immature 13, cortical 17, mature 5) and validation cohort (n=99; immature 45, cortical 44, mature 10). Total RNA seq was done in the discovery cohort. Clinical and prognostic relevance of lesions identified in the discovery cohort were studied in validation cohort. On RNAseq, a total of 2,318 genes were most differentially expressed between the 3 subtypes. In immature T-ALL cases, transcription factors controlling early hematopoiesis (MEF2C, TP63, HHEX, RUNX2, HOXA10, HOXA9, RUNX1T1 and ZBTB16) were highly expressed. LYN, a tyrosine kinase gene was also highly expressed. BAALC and MN1 genes, previously reported in acute myeloid leukemia, but not emphasized in reported genomics studies in the West, were overexpressed. Interestingly, LMO2 was overexpressed in the absence of significant overexpression of LYL1, a gene reported to be critical for oncogenic functions of Lmo2. In cortical T-ALL, the cortical thymocyte-defining CD1A gene was overexpressed. Homoebox domain genes NKX2-1, TLX1, TLX3, involved in T-cell development were overexpressed. In addition, FAT1, FAT3, EREG, CD1C, AKAP-2, IL-4, PRTG, TCL-6, ZP1 and TRAV genes were overexpressed. On examining CD1a+/sCD3+ and CD1a+/sCD3- groups, we found that in contrast to reported literature, TLX1 was highly expressed in the former group and TLX3 in the latter. In mature T-ALL, APC2, BCL3, CCR4, CDKN2A, EML4, HIST1H4G, HIST2H2B, NCOR2, ST20 and TRAV22 genes were overexpressed. Conventional drivers of T-ALL leukemogenesis - LYL1, TAL1 and LMO1 were not differentially expressed in our patients suggesting a difference between Indian and Western T-ALL patients. The clinical significance of expression of MEF2C, BAALC, HHEX, LYL1, TAL1 and FAT1 genes was studied in validation cohort by real-time PCR. High expression of MEF2C and BAALC gene was associated with immature immunophenotype (IPT) as compared to cortical and mature IPT (p=0.009; 0.008, respectively). Patients with ETP-ALL had higher expression of MEF2C and BAALC gene (p=0.018; 0.014, respectively). High MEF2C and BAALC expression were associated with CD34 positivity (p=0.004; 0.032, respectively). High BAALC expression was also associated with aberrant expression of the myeloid markers (p=0.021). Although not statistically significant, high HHEX expression was associated with immature IPT (p=0.072) and ETP-ALL IPT (p=0.06). Although LYL1 expression did not correlate with immature IPT, ETP-ALL was associated with higher LYL1 expression (p=0.039). Higher expression of FAT1 gene was associated with cortical IPT (p=0.026). Low XIST expression was more frequently associated with non-ETP-ALL IPT (p=0.017). TAL1 expression did not correlate with IPT. On survival analysis, only MEF2C gene expression was associated with EFS and OS. Compared to low MEF2C expressors, patients with high MEF2C had a lower event free survival (EFS) (73.58% vs 51.38%, p=0.01). High expression was found to be a significant predictor of overall survival (OS) (81.11% vs 96.43%, p=0.012). MEF2C emerged as driving oncogene in our T-ALL patients which can be used as predictor of EFS and OS. Expression of BAALC, HHEX, LYL1, TAL1, FAT1 and XIST genes did not correlate with EFS and OS. In this first study correlating T-ALL IPT with transcriptome profile in Indian T-ALL patients, we noted interesting differences from the West, potential subjects of further study. MEF2C gene expression emerged as the predictor of poor EFS and OS in T-ALL patients. Lastly, increased expression of LYN gene in our patients suggests that our immature T-ALL patients may benefit from tyrosine kinase inhibitors targeting Lyn. Disclosures No relevant conflicts of interest to declare.

3084 - Distinct and Overlapping Function for Scl, Lmo2 and Ldb1 in Regulating the Divergence of Hematopoietic and Cardiac Fates

Lmo2 and Ldb1 are non-DNA binding components of a multi-factor complex containing Scl/Tal1, the master transcription factor that regulates hematopoietic fate commitment during embryogenesis. Scl has been shown to be critical for the activation of the hematopoietic transcription program in hemogenic endothelium, and repression of cardiac transcription program in endothelium and endocardium, and hence responsible for the divergence of these mutually exclusive mesodermal fates. However, the specific functions of Lmo2 and Ldb1 in this complex, especially in the repressive function, are poorly understood. Here, we employ a combination of germline and conditionally-deleted Scl, Lmo2 and Ldb1 mouse model to study the dynamic roles of each complex member. Our data suggests that similar to Scl, both Lmo2 and Ldb1 are critical for primitive and definitive erythropoiesis. However, whereas the deletion of Scl completely ablates HSPC specification, the presence of residual CD41+cKit+ HSPCs in Lmo2 and Ldb1 knockout embryos suggests that they may have partially redundant roles during HSPC specification. Lmo2, similar to Scl, is critical for the repression of cardiac fate in hemogenic endothelium and endocardium, but becomes dispensable before Scl for continued cardiac repression in the endocardium. Ldb1-/- embryos lack heart and instead show accumulation of CD31+Pdgfra+ cells in the rostral half. Moreover, hemogenic tissues of Ldb1 mutants do not show accumulation of cardiogenic progenitors but exhibit an expansion of CD31+cKit+ endothelial cells, suggesting that Ldb1 critically regulates the activation of both hematopoietic and cardiac transcriptional program in the endothelium. ChIP-seq revealed that Lmo2 predominantly acts through Scl-bound enhancers. Ldb1 also has an Scl independent function in activating cardiac genes, as well as a distinct, but Scl-dependent, function in regulating genes associated with polycomb-repression such as Ezh2 and Suz12. Our data demonstrates the dynamic and temporal interactions as well as individual functions between the members of Scl complex during the divergence of hematopoietic and cardiac lineages.

A comprehensive function analysis of LMO2 in different breast cancer subtypes.

Breast cancer is the most common invasive cancer in women worldwide, and can be subdivided into Luminal A, Luminal B, Her2, and Basal subtype (the PAM50 subtyping system). The lmo2 gene was traditionally recognized as a proto-oncogene in hematopoietic system but its functions in breast cancers remained largely unclear. Based on the Cancer Genome Atlas (TCGA) breast cancer dataset, herein we found that the significantly LMO2-correlated genes in normal or malignant samples were enriched in rather divergent cellular pathways, suggesting the function complexity of LMO2 in breast tissues. Moreover, high LMO2 expression level was found to predict a shorter patient survival in Luminal A type whereas a better outcome in Her2 type. Correspondingly, LMO2 also revealed function diversities in different PAM50 subtypes. In Luminal A type, the LMO2 related genes were primarily enriched in cancer-promoting pathways, including VEGF production, stemness, PPAR signal pathways, MAPK cascade and cell cycle regulation. In Her2 type however, the LMO2 related genes lacked the enrichment on most of the generally cancer-related pathways and were particularly enriched in negative regulation of ErbB pathway as well as MAPK cascade, suggesting a potentially anti-oncogenic role of LMO2 on this subtype. Taken together, this study drew a comprehensive overview of divergent functions of LMO2 on breast cancers, provided additional evidence for the function complexity of LMO2 in solid tumors and suggested the potential usage of LMO2 as a PAM50 subtype dependent biomarker for breast cancer clinic in the precision medicine era.

The dual function of LMO2 in driving erythroid cell fate

The dual function of LMO2 in driving erythroid cell fate

LMO2 promotes tumor cell invasion and metastasis in basal-type breast cancer by altering actin cytoskeleton remodeling.

LMO2 is traditionally recognized as a pivotal transcriptional regulator during embryonic hematopoiesis and angionenesis, and its ectopic expression in T lymphocyte progenitors is closely correlated to the onset of acute T lymphocytic leukemia. However, recently studies revealed complicated expression features and dual functions of LMO2 on tumor behaviors in a variety of cancer types, including breast cancers. Basal-type breast cancer is one of the breast cancer subtypes and a prognostically unfavorable subtype among all breast cancers. Herein we found that in basal-type breast cancer specifically, high LMO2 expression was positively correlated with lymph node metastases in patients, promoted tumor cell migration and invasion and increased distant metastasis in SCID mice. Moreover, the novel function of LMO2 was achieved by its predominantly cytoplasmic location and interaction with cofilin1, which is a critical regulator in actin cytoskeleton dynamics. These findings suggest a subtype-dependent role of LMO2 in breast cancers and the potential of LMO2 as a subtype-specific biomarker for clinical practice.

LMO2 attenuates tumor growth by targeting the Wnt signaling pathway in breast and colorectal cancer.

The proto-oncogene LIM-domain only 2 (lmo2) was traditionally considered to be a pivotal transcriptional regulator in hematopoiesis and leukemia. Recently, the cytosolic localization of LMO2 was revealed in multiple epithelial tissues and a variety of solid tumors. However, the function of LMO2 in these epithelia and solid tumors remains largely unclear. The Wnt signaling pathway is a crucial determinant of development, and abnormalities in several key segments of this pathway contribute to oncogenesis. The current study demonstrated that LMO2 participates in the regulation of canonical Wnt signaling in the cytoplasm by binding to Dishevelled-1/2 (DVL-1/2) proteins. These interactions occurred at the PDZ domain of Dishevelled, and LMO2 subsequently attenuated the activation of the key factor β-catenin in the canonical Wnt signaling pathway. Meanwhile, significantly decreased expression of LMO2 was detected in breast and colorectal cancers, and the downregulation of LMO2 in these cells increased cell proliferation and reduced apoptosis. Taken together, the data in this study revealed a novel crosstalk between LMO2 and the Wnt signaling pathway during tumorigenesis and suggested that LMO2 might be a tumor suppressor in certain solid tumors, in contrast to its traditional oncogenic role in the hematopoietic system.

LMO2 blocks the UBA6-USE1 interaction and downstream FAT10ylation by targeting the ubiquitin fold domain of UBA6

In eukaryotic cells, the post-translational modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is the most common trigger for protein degradation and is involved in the regulation of a wide range of biological processes. FAT10 (HLA-F-adjacent transcript 10), which belongs to the UBL family, is activated specifically through the UBA6-USE1 cascade and targets substrates covalently for 26S proteasomal degradation. LMO2 is a well-recognized transcriptional regulator in hematopoietic and endothelial systems; however, it is predominantly located in the cytoplasm of epithelium-derived cells. The current study revealed that LMO2 protein interacted with the E1 ubiquitin-activating enzyme UBA6 at the C-terminal ubiquitin fold domain (UFD), which mediates the recognition and recruitment of the E2-conjugating enzyme USE1. Functionally, the LMO2-UBA6 interaction disturbed the interaction between UBA6 and USE1 and led to the decline of the overall cellular FAT10ylation level as well as the FAT10ylation and degradation of a known FAT10 substrate p62. Taken together, this study revealed a novel function of LMO2 involving in the regulatory hierarchy of UBA6-USE1-FAT10ylation pathway by targeting the E1 enzyme UBA6.

LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding.

LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.

LMO2 at 25 years: a paradigm of chromosomal translocation proteins.

LMO2 was first discovered through proximity to frequently occurring chromosomal translocations in T cell acute lymphoblastic leukaemia (T-ALL). Subsequent studies on its role in tumours and in normal settings have highlighted LMO2 as an archetypical chromosomal translocation oncogene, activated by association with antigen receptor gene loci and a paradigm for translocation gene activation in T-ALL. The normal function of LMO2 in haematopoietic cell fate and angiogenesis suggests it is a master gene regulator exerting a dysfunctional control on differentiation following chromosomal translocations. Its importance in T cell neoplasia has been further emphasized by the recurrent findings of interstitial deletions of chromosome 11 near LMO2 and of LMO2 as a target of retroviral insertion gene activation during gene therapy trials for X chromosome-linked severe combined immuno-deficiency syndrome, both types of event leading to similar T cell leukaemia. The discovery of LMO2 in some B cell neoplasias and in some epithelial cancers suggests a more ubiquitous function as an oncogenic protein, and that the current development of novel inhibitors will be of great value in future cancer treatment. Further, the role of LMO2 in angiogenesis and in haematopoietic stem cells (HSCs) bodes well for targeting LMO2 in angiogenic disorders and in generating autologous induced HSCs for application in various clinical indications.

Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL

AbstractLmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell–like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.

Identification of LMO2 transcriptome and interactome in diffuse large B-cell lymphoma

AbstractLMO2 regulates gene expression by facilitating the formation of multipartite DNA-binding complexes. In B cells, LMO2 is specifically up-regulated in the germinal center (GC) and is expressed in GC-derived non-Hodgkin lymphomas. LMO2 is one of the most powerful prognostic indicators in diffuse large B-cell (DLBCL) patients. However, its function in GC B cells and DLBCL is currently unknown. In this study, we characterized the LMO2 transcriptome and transcriptional complex in DLBCL cells. LMO2 regulates genes implicated in kinetochore function, chromosome assembly, and mitosis. Overexpression of LMO2 in DLBCL cell lines results in centrosome amplification. In DLBCL, the LMO2 complex contains some of the traditional partners, such as LDB1, E2A, HEB, Lyl1, ETO2, and SP1, but not TAL1 or GATA proteins. Furthermore, we identified novel LMO2 interacting partners: ELK1, nuclear factor of activated T-cells (NFATc1), and lymphoid enhancer-binding factor1 (LEF1) proteins. Reporter assays revealed that LMO2 increases transcriptional activity of NFATc1 and decreases transcriptional activity of LEF1 proteins. Overall, our studies identified a novel LMO2 transcriptome and interactome in DLBCL and provides a platform for future elucidation of LMO2 function in GC B cells and DLBCL pathogenesis.

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

LMO2 is a transcription regulator involved in human T-cell leukemia, including some occurring in X-SCID gene therapy trials, and in B-cell lymphomas and prostate cancer. LMO2 functions in transcription complexes via protein-protein interactions involving two LIM domains and causes a preleukemic T-cell development blockade followed by clonal tumors. Therefore, LMO2 is necessary but not sufficient for overt neoplasias, which must undergo additional mutations before frank malignancy. An open question is the importance of LMO2 in tumor development as opposed to sustaining cancer. We have addressed this using a peptide aptamer that binds to the second LIM domain of the LMO2 protein and disrupts its function. This specificity is mediated by a conserved Cys-Cys motif, which is similar to the zinc-binding LIM domains. The peptide inhibits Lmo2 function in a mouse T-cell tumor transplantation assay by preventing Lmo2-dependent T-cell neoplasia. Lmo2 is, therefore, required for sustained T-cell tumor growth, in addition to its preleukemic effect. Interference with LMO2 complexes is a strategy for controlling LMO2-mediated cancers, and the finger structure of LMO2 is an explicit focus for drug development.

An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions

The LIM-domain protein LMO2 is a T-cell oncogenic protein first recognized by gene activation through chromosomal translocations, but it is also responsible for leukaemias arising as secondary, adverse effects in an X-SCID gene therapy trial. There are no specific reagents currently available to analyse the LMO2 multiprotein complex or to combat LMO2-dependent leukaemias. Accordingly, we have isolated an anti-LMO2 single chain Fv antibody fragment to determine if intracellular interference with LMO2-protein complexes can avert LMO2-dependent functions in normal and cancer settings. The anti-LMO2 single chain Fv, obtained using Intracellular Antibody Capture (IAC) technology, is specific for LMO2 among the LIM-only protein family and binds LMO2 through the third and fourth LIM fingers. Using vector-mediated expression of anti-LMO2 scFv, we show inhibition of Lmo2-dependent erythropoiesis but not endothelial development. We also demonstrate inhibition of Lmo2-dependent leukaemia in a mouse T-cell tumourigenesis transplantation assay with retroviral-mediated expression of anti-LMO2 scFv. Our studies establish that interference with the LMO2 multiprotein complex inhibits both normal and tumourigenic roles. The antibody fragment is a tool for dissecting LMO2 function in haematopoiesis and leukaemia and is a lead for development of therapeutics against LMO2-dependent T-ALL.

The LIM-domain protein Lmo2 is a key regulator of tumour angiogenesis: a new anti-angiogenesis drug target.

The growth of solid tumours requires a blood supply provided by re-modeling of existing blood vessel endothelium (angiogenesis). Little is known about transcription regulators which are specific for the control of tumour angiogenesis. The proto-oncogene LMO2 encodes a LIM domain transcription regulator which controls angiogenesis during mouse embryogenesis where it regulates remodelling of the capillary network into mature vessels. We now show that Lmo2 expression is augmented in tumour endothelium such as mouse thymomas and human lung tumours. The functional significance of this Lmo2 expression was assessed in teratocarcinomas induced in nude mice by subcutaneous implantation of Lmo2-lacZ targeted ES cells. CD31-positive, sprouting endothelium of ES-cell origin occurred in teratocarcinomas from heterozygous Lmo2-lacZ ES cells but none occurred from null Lmo2-lacZ ES cells. Therefore, in this model Lmo2 is an obligatory regulator of neo-vascularization of tumours. These data suggest that LMO2 function may be a drug target in cancer and other conditions characterized by neo-vascularization.

Identification of genes potentially involved in LMO2-induced leukemogenesis

The most common translocations in childhood T cell acute lymphoblastic leukemias involve the LMO2 locus on chromosome 11p13 and cause ectopic expression of the LMO2 gene in thymocytes. Transgenic mice with enforced expression of LMO2 in their thymocytes develop T cell leukemias thus demonstrating the role of LMO2 in leukemogenesis. The physiologic and leukemogenic functions of LMO2 are mediated through its transcriptional regulatory activities, but the identity of the target genes is completely unknown. In this report, we have used cDNA representational difference analysis (cDNA-RDA) to identify genes that are over-expressed and are likely to play a role in the LMO2 induced leukemias. cDNA-RDA was performed using very small amounts of mRNA pool (from 1 microg of total RNA) to reverse transcribe the cDNAs from leukemic cells or normal thymocytes. The cDNA-RDA led to the isolation of nine distinct clones that were specifically overexpressed in the leukemic cells. Sequence analysis revealed that five of the nine clones had identity or homology to known genes that are known to play a role in the pathogenesis of leukemias or other cancers. Three clones had no significant homology to any known genes and thus represent novel candidate genes. Our study demonstrates that cDNA-RDA using very small amounts of total RNA is a highly efficient method to identify novel genes that may play a role in leukemogenesis.