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Hematopoiesis is a very organized process which is tightly controlled by various transcription factors.
Hematopoiesis is one of the best understood developmental systems for studying cell self-renew and differentiation. The hematopoietic stem cells (HSCs) differentiate into a range of specific progenitor cells which further give rise to all the mature blood cell types. This process is tightly controlled by various lineage-determining transcription factors. While misexpression of these transcription factors commonly leads to the development of leukaemia.
One protein with a crucial role in hematopoiesis development is LIM (Lin-1, Isl-1, Mec-3) domain only 2 (LMO2), a member of the LIM-only zinc finger protein family. Mice lacking Lmo2 die around embryonic day 10.5 because of a complete failure of yolk sac (primitive) erythropoiesis(1). Further studies of chimeric mice made by injecting Lmo2-/- embryonic stem (ES) cells have shown that Lmo2 is also required for adult (definitive) hematopoiesis(2). Following differentiation of HSCs, Lmo2 expression is maintained in erythroid cells but is down-regulation in T lymphocytes where aberrant expression of LMO2 results in T-cell leukemia(3, 4). In addition, the activation of LMO2 expression as a consequence of retrovial insertion was the cause of T-ALL in four patients undergoing gene therapy for X-linked severe combined immunodeficiency(5, 6).
The regulation of LMO2 expression therefore has implications for both haematopoiesis and leukaemogenesis. A better understanding of how aberrant expression of LMO2 results in T-cell leukemias will provide rational approaches for identifying new therapeutic strategies.
1, LMO family
The LIM domain is a highly conserved cysteine-rich zinc finger-like motif that was present in certain homeodomain transcription factors, some kinases and cytoskeletal proteins, and acts as a docking site for the assembly of multiprotein complex(7). The LIM-domain-only (LMO) subclass of LIM proteins is a family of nuclear transcription co-regulators that are characterized by the exclusive presence of two tandem LIM domains and no other functional domains. The central role of LMO proteins was so far seen as nuclear transcriptional co-regulators, mediating protein–protein interactions of various transcription factors or chromatin modeling proteins, which may have either positive or negative effects on gene transcription. To date, four LMO proteins (LMO1-LMO4) have been identified. LMO1 and LMO2 were found to be overexpressed as a result of chromosomal translocations in patients with T cell acute lymphoblastic leukaemia (T‑ALL), LMO3 is considered to be a neuroblastoma-associated oncogene, and LMO4 was originally identified as a human breast cancer auto-antigen(8).
2, LMO2 in erythropoiesis
LMO2 plays a very important role for erythroid differentiation. Homozygous inactivation of the Lmo2 gene in mice led to the complete absence of yolk sac erythropoiesis and early embryonic lethality(1). Further, the erythroid differentiation was completely blocked in both Lmo2–/– ES cells and wild-type ES cells transduced with an anti-LMO2 single-chain antibody(1) (9). In contrast, it has been reported that LMO2 overexpression inhibits erythroid differentiation of erythroid progenitor cell line G1E-ER-GATA-1(10). Since LMO2 could have opposite roles in early- and late-stage erythropoiesis, it makes the study of regulation of LMO2 expression and the interaction with other transcription factors much more important.
Is tightly regulated by a network of transcription factors including LMO2 itself, SCL, GATA1, and ETS factors.
In erythroid cells, Lmo2 functions as a bridging molecule assembling a multiprotein transcriptional complex that includes hetero-dimers of E proteins (E47 or HEB) and Scl or Lyl1, as well as Gata1 and Ldb1. The multimeric GATA-1/SCL/Ldb1/E2A/LMO2 complex binds to closely spaced GATA and E-box binding motifs and has been associated with the activation of erythroid genes, such as glycophorin A and the a-globin locus(11, 12).
3, LMO2 in T cell leukemia
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy which occurs both in children and adults. Children have an overall survival rate of approximately 80% while adults only around 30%-40%, with a significant proportion of patients succumbing to relapsed or resistant disease.(13, 14) Approximately 9% of T-ALL cases exhibit abnormally increased LMO2 expression.(13, 15)
The most common translocation in childhood T-ALL involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus.
LMO2 was subsequently identified at several different human T‑ALL chromosomal translocations: t(11;14)(p13;q11) and t(7;11)(q35;p13), as well as a cryptic deletion (del(11)(p12p13))(3, 16, 17). These chromosomal abnormalities that involve LMO2 account for ~9% of paediatric cases of T-ALL(17), and all result in the aberrant overexpression of LMO2. The t(11;14)(p13;q11) and t(7;11)(q35;p13) translocations both result in T cell receptor genes, the δ‑chain gene at chromosome 14q11 and the β‑chain gene at chromosome 7q35, being fused upstream of LMO2 at chromosome 11p13. By contrast, the cryptic deletion is postulated to remove a negative regulatory element and to thereby increase expression. (8)
LMO2‑induced leukaemias have also arisen as an unfortunate side effect of gene therapy trials for X‑linked severe combined immunodeficiency (SCID‑X1) (BOX 2). In these cases it was found that a retroviral copy of the replacement interleukin‑2 receptor-γ (IL2RG) gene had inserted in or close to LMO2. Thus, in T‑ALL caused by LMO2 translocations or gene therapy, the insertion of genes that are highly expressed in T cells in the LMO2 locus drives the transcriptional upregulation of LMO2 in T cells, which in turn contributes to T‑ALL. (8)
The protein LMO2 is expressed at high levels in hematopoietic stem cells, multipotent progenitor cells, and in early T-cell progenitors, but is downregulated at the DN2 stage and not expressed in subsequent T-cell progenitor cells or mature T cells.(18-20) Overexpression of LMO2 causes a specific block at the DN3 stage, which is also the point of b selection, where T-cell progenitors with productively rearranged T-cell receptors proliferate and are blocked from apoptosis.(19, 21, 22)
How LMO2 contributes to T cell transformation remains unclear. Lyl1 is required for the oncogenic effects of Lmo2 in developing T cells, and it is required for growth of ETP-ALL cell lines(23). Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL.(13)
In review: (24)
Mentioned possible LMO2 target genes: protein 4.2 (p4.2), TALLA1, RALDH2, CKIT and EKLF
TALLA1 is a member of the transmembrane 4 superfamily and normally expressed in neurons, certain vascular endothelial cells and certain epithelial cells but not in any hematopoietic cells including T cells. Its highly frequent ectopic expression and 100% concordance with TAL1 in various T-ALL cell lines indicate TALLA1 as a highly specific tumor marker of T-ALL. TALLA1 expression can be strongly induced by coexpression of TAL1 and LMO1 or LMO2 in a TALLA1-negative T-ALL cell line.(25, 26)
RALDH2 (retinaldehyde dehydrogenase 2) is identified as one target of LMO2 in T-ALL leukemogenesis. RALDH2 is an enzyme converting retinal to retinoic acid. While retinoic acid has been shown the ability to inhibit activation-induced apoptosis of T cells, the ectopically expressed RALDH2 inhibits apoptosis of T cells by generating retinoic acid (27, 28). LMO2 together with TAL1 and GATA3 form a transcriptional complex which binds to a GATA site in a cryptic promoter in the second intron of the RALDH2 gene and regulates its transcription(24, 29).
The Hhex (hematopoietic-expressed homeobox), also known as PRH (proline-rich homeodomain), is a transcription factor that contains the homeodomain. The Hhex is expressed in early haematopoietic progenitors of all lineages except T-cell lineages. Chimaeric mice with only homozygous null for Hhex in the lymphoid lineages have shown that Hhex is essential for B-cell function and development but does not affect T-cell development. Overexpression of Hhex in mouse bone marrow results in death of all other haematopoietic cells but an aggressive T-cell lymphoma in bone marrow, indicating that Hhex may function as a T-cell lineage oncogene.(30)
Hhex is overexpressed ten fold in LMO2-transgenic thymocytes. Both enforced expression of LMO2 and Hhex in thymocytes result in abnormal expression of Kit, CD25 and CD5, suggesting that Hhex could be an important downstream target of LMO2. (21, 31) (32) Reporter assays suggested that both the Hhex promoter and enhancer were active in T-ALL. Chromatin immunoprecipitation (ChIP) using antibodies against LMO2, LDB1, LYL1, and GATA3 showed enrichment for the promoter and enhancer of Hhex suggesting specific occupancy.(33)
DeltaEF1 is a widely expressed transcription factor belonging to the zinc finger-homeodomain family. Knock out of DeltaEF1 severely impaired T-cell development by arresting early T-cell differentiation, implicating a critical function of DeltaEF1 in hematopoiesis especially in T lineage. Transgenic mice study showed that knock out of deltaEF1 gene or overexpression of LMO2 carried out similar phenotype of T-cell development arrest, implying a possible functional relationship between these two genes. It has been reported that ectopically expressed LMO2 targeted to DeltaEF1 promoter and inhibited its expression by interacting with GATA3, a T-lineage-specific transcription regulator that positively regulated DeltaEF1. Meanwhile, LMO2 bound to the activation domain N-terminal zinc finger domain (NZF) of DeltaEF1 protein and inhibited the positive regulatory function of DeltaEF1 on its targets. Taken together, ectopically expressed LMO2 impaired the function of DeltaEF1 in both transcriptional and protein levels and identified DeltaEF1 as a novel pathogenic target of LMO2 in T-cell leukemia.(34)
Cell line 32080 comes from CD2-Lmo2 transgenic mice with T-ALL. It can switch between two T-cell progenitor stages, the intermediate CD8 single positive stage and the double positive stage, and has an obvious variegated pattern of CD4 expression. Knockdown of LMO2 in this cell line caused decreased CD4 expression, while increased LMO2 by inhibiting NOTCH1 activity can increase CD4 expression, suggesting that LMO2 promotes CD4 expression. Chromatin immunoprecipitation shows that decreased CD4 expression in 32080 cell line is companied by decrease of H3K4me3, H3K9me2 and H3K27me3 and increased H3K9me3 and H3ac, which indicates LMO2 induces T-cell leukemia with epigenetic deregulation of CD4.(19)
4, LMO2 regulator elements and isoforms
During the differentiation of HSCs, Lmo2 expression is down-regulated in T lymphocytes, while aberrant expression of LMO2 will result in T-cell leukemias. Thus, appropriate transcriptional control of LMO2 is crucial for the formation and subsequent behavior of blood cells. Understanding the molecular mechanisms controlling the expression of LMO2 will provide valuable information for the transcriptional control of hematopoietic development. (35)
According to NCBI AceView genes database (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html)(36), LMO2 gene has 7 probable alternative promoters. Its transcription produces 13 different mRNAs (11 alternatively spliced variants and 2 unspliced forms), among which 7 variants encode good proteins. The regulation system should be very complicated. Expression of the leukemia oncogene LMO2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors(37). So far 4 promoters, 8 enhancers, 1 negative regulatory element and 2 isoforms have been studied.
The proximal promoter is present in the vicinity of exon 3, which was functional in hematopoietic progenitor and endothelial cell lines. Its activity was dependent on conserved Ets sites bound by Fli1, Ets1, and Elf1 (38). The distal promoter is located nearly 25 kb upstream, which is active in the fetal liver and specific T-cell acute lymphoblastic leukemia (T-ALL) cell lines (39). The intermediate promoter locates just before exon 2, which was found by using ChIP with antibodies targeting histone modification marks coupled with microarray technology to discover regulatory sequences active in T-ALL primary cells where high levels of LMO2 were present in the absence of any translocation involving the LMO2 locus (35). FLI1 and ERG can bind to the intermediate and activate LMO2 expression. Further on, LMO2 together with FLI1 and ERG bind to a powerful enhancer element in HHEX/PRH, thus collaborate to form a key regulatory subcircuit of the wider transcriptional networks driving the development of T-ALL (35).
However, none of these promoters on their own displayed robust expression when tested in transgenic mice (35, 38), which led to the identification of eight enhancer elements dispersed over 100 kb that could recapitulate the expression of Lmo2 in normal haematopoiesis. The eight regions (-90, -75, -70, -64, -25, -12, -1, and -7) significantly augmented the endothelial staining of pPLacZ and/or induced LacZ expression in several additional tissues, such as tail, apical ridges of the limbs, brain, and potentially FL. (41)
The regulatory machinery of the LMO2 oncogene includes both positive and negative elements. Suppression of LMO2 in T cells mediated by a negative element that maps to a region consistently removed from the gene upon chromosomal translocation in T cell leukemias. Further studys delineate the repressor region to a 205 bp fragment from −2120 to −1915. The distal LMO2 repressor element is located within this 205 bp region but does not conform to a known consensus binding site.(42)
The proximal promoter and the distal promoter encode a same product LMO2-L. A group later cloned a new transcript from human adult kidney, termed Lmo2-c. Lmo2-c has the promoter of its own and encodes a shorter isoform, termed LMO2-S, which has 14 amino acids missed in the N-terminal region compared to LMO2-L. It was driven by a novel promoter which starts at the end of exon 3. Both GATA-1 and PU.1 mediate LMO2-c transcription through binding to its promoter region. PU.1 suppresses GATA-1-mediated stimulation of LMO2-c promoter through interacting with GATA-1 on the GATA-1 binding site. (40)
5, LMO2 as a cancer target molecule
The oncogenic functions of LMO2 in T‑ALL have motivated efforts to generate LMO2‑targeted agents as potential anticancer therapeutics. Based on the known molecular mechanism of Lmo2 in T-ALL, Dr. McCormack proposed three main therapeutic ways for treating LMO2‑induced T‑ALL: by targeting the Lmo2-contianing transcription complex, targeting critical Lmo2 downstream mediators of self-renewal or targeting the precancerous stem cell niche.
So far, two strategies have been developed by the Rabbitts laboratory. The first is an intrabody which is an intracellular single-chain antibody that binds to LMO2 specifically(9). The second is an eight-amino acid peptide aptamer which contains a C‑X‑X‑C motif that can bind to the carboxy-terminal LIM domain of LMO2(43). Both approaches could block the differentiation of erythroid cells in an in vitro assay, and could inhibit the proliferation of neoplastic T cells in a mouse explant model of LMO2‑induced T‑ALL. These studies indicate that molecular targeting of LMO2 may be an effective strategy for treating LMO2‑induced T‑ALL. However, given that LMO2 is an important factor for normal haematopoietic stem cell and erythrocyte development, the delivery of reagents into specific cell populations such as DN3 cells is required before anti‑LMO2 reagents could be used in the clinic.