Activation of the T-Cell Oncogene LMO2 after Gene Therapy for X-Linked Severe Combined Immunodeficiency
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《新英格兰医药杂志》
The emerging field of gene therapy promises to yield exciting new treatments for a range of illnesses. Most forms of gene therapy use viral delivery mechanisms, most commonly retroviruses,1 and one condition in which this approach has corrected a genetic defect is X-linked severe combined immunodeficiency (SCID).2 X-linked SCID is characterized by the lack of specific types of lymphocytes (Figure 1A and Figure 1B)7,12 owing to the absence of the common subunit of the interleukin-2 receptor (IL2Rc). This subunit of the interleukin-2 cytokine receptor is a signaling molecule that is a component of receptors for several other lymphoid-specific growth factors, and in its absence, mature lymphocytes (both B and T cells) cannot develop (Figure 1B). The resulting impairment of immune responses allows insuperable opportunistic infections, which usually cause death before the age of two years. However, in some cases the immune defect can be corrected by allogeneic bone marrow transplantation.
Figure 1. Normal (Panel A), Defective (Panel B), and Malignant (Panels D and E) Hematopoiesis in Persons with and without X-Linked Severe Combined Immunodeficiency (SCID), and Correction of the Lymphocyte Defect with the Use of Gene Therapy (Panel C).
In Panel A, normal hematopoiesis involves differentiation of pluripotential hematopoietic stem cells expressing the CD34 surface protein (CD34+) into two main committed progenitors: common myeloid and common lymphoid progenitors.3,4,5,6 Common myeloid progenitors can differentiate into all cells of the myeloerythroid lineage (granulocytes, macrophages, megakaryocytes, erythroid cells), and common lymphoid progenitors can differentiate into T cells, B cells, and natural killer cells. Boys with X-linked SCID are deficient in the common chain of the interleukin-2 receptor (IL2Rc), causing failure of normal lymphopoiesis (Panel B).7 Gene therapy was used to correct the defect in X-linked SCID (Panel C). CD34+ hematopoietic stem cells were infected ex vivo with a retrovirus encoding IL2Rc, and transduced autologous cells were reimplanted. Since the hematopoietic stem cells are progenitors of all hematopoietic cells, the transduced cells contribute cells carrying the integrated IL2Rc-retroviral DNA to all lineages, including the lymphocyte lineage, thereby correcting the immunodeficiency.2 The proportion of cells carrying the inserted retrovirus in the lymphoid lineages far outweighs that in other lineages. Unfortunately, T-cell leukemia developed in two of the patients successfully treated in this way (Panel D).8 In both children, the T-cell acute lymphoblastic leukemia (T-cell ALL) arose in cells in which the retroviral integration site was within or very close to the T-cell oncogene LMO2.9 This enforced LMO2 gene activation effectively mimics the molecular event that led to the discovery of the LMO2 gene — namely, chromosomal translocation in T-cell leukemia.10,11 In T-cell ALL involving the chromosomal translocation t(11;14)(p13;q11) or t(7;11)(q35;p13), activation of LMO2 occurs by means of the break point of these translocations, near a T-cell receptor gene, and its activation leads to leukemia (Panel E).
In a recent successful trial of gene therapy in Paris, CD34+ hematopoietic stem cells from patients with X-linked SCID were cultured ex vivo and transduced with a defective retroviral vector, based on the Moloney murine leukemia virus that carried the human IL2Rc gene.2 These transduced cells were reintroduced into the patients, who were then able to produce T cells and natural killer cells (Figure 1C). Levels of T cells in the blood reached normal values within a few months and have remained so for several years, although low numbers of natural killer cells persist.2 This result is the first clear demonstration of a clinical benefit of gene therapy and is a major step in the correction of inherited diseases, especially those involving deficiencies of hematopoietic cells.
A Major Adverse Effect of Viral Gene Therapy
Although gene therapy for X-linked SCID is a most important advance, it has sadly also uncovered a major adverse effect, which vividly demonstrates an intrinsic danger of retroviral vectors. Retroviral vectors are used in gene therapy because they can stably insert themselves into the genome of host cells. However, retroviruses of the type used in the French trial can also enhance the expression of cellular genes that surround the site of insertion of the vector through insertional mutagenesis.13 The possibility that the retroviruses used in gene therapy may cause insertional mutagenesis, thereby evoking untoward side effects, has been discussed for many years.14,15 Moreover, experiments in mice have shown that retroviral insertional mutagenesis causes leukemia and have yielded many candidate leukemia oncogenes.13,16,17,18 The risk of oncogenic insertional mutagenesis in humans as a consequence of gene therapy with the use of a retroviral vector was, however, deemed to be low. But the outcome of the X-linked SCID trial has shown that this risk is a major problem because, although 9 of 10 children treated with the IL2Rc-rescue protocol are in remission (long-term follow-up is essential before they can be considered to be cured), a T-cell acute leukemia-like syndrome has developed in 2 of the boys (Figure 1D).8,9 This event has been attributed to the integration of the retrovirus into a known T-cell oncogene, LMO2,9 in the deregulated T-cell clones (Figure 2), thereby contributing to the development of T-cell leukemia by causing aberrant expression of LMO2.
Figure 2. Molecular Architecture of the LMO2 Gene and Break Points for Retroviral Insertion or Chromosomal Translocations.
The LMO2 gene is located on the short arm (p) of human chromosome 11 at band 13 (11p13). The gene has two transcriptional promoters19 and comprises six exons, of which exons 4, 5, and 6 code for protein. Chromosomal translocations involving LMO2 are found in T-cell acute leukemias, and the chromosome 11p13 break point can be paired with either the T-cell receptor gene (TCR) on chromosome 14 (band q11) or the T-cell receptor gene (TCR) on chromosome 7 (band q35).20 All known chromosomal translocations occur upstream of one or both of the LMO2 promoters.21 The same feature is true of the X-linked severe combined immunodeficiency retroviral (MFGc) insertion sites associated with leukemia, which were found either just ahead of LMO2 exon 1 or between exons 1 and 2.9 In both circumstances the LMO2 protein product is identical before and after the genetic abnormality has occurred, and it is the enforced expression of LMO2 that influences T-cell development and leukemia. IL2Rc denotes interleukin-2 receptor common subunit, Met methionine protein translation-initiation codon, and stop protein-translation stop codon.
LMO2, a Quintessential Chromosomal Translocation Oncogene
The occurrence of T-cell leukemia after forced expression of the LMO2 gene by a retrovirus is not surprising, given that LMO2 is involved in spontaneous cases of T-cell leukemia.10,11 LMO2 is found at the break points of the t(11;14)(p13;q11) or the t(7;11)(q35;p13) translocations in T-cell acute lymphoblastic leukemia (T-cell ALL)20,22 (Figure 2) and encodes a LIM-only protein (LMO2), which acts as a bridging molecule in transcription-factor complexes (Figure 3). LMO2 is present in the progenitors of all hematopoietic lineages before its expression is down-regulated in all except the erythroid lineage.38 In T cells, down-regulation of the protein appears to be crucial, since T-cell leukemia develops in mice that constitutively express Lmo2 in the thymus.39,40 The overt leukemia is preceded by an accumulation of immature thymic T cells, indicating that deregulated Lmo2 increases susceptibility to leukemia by blocking the differentiation of T cells.36,41,42 Only T-cell leukemia develops in transgenic mice, even though Lmo2 is expressed in many tissues, implying that this gene is specifically a T-cell oncogene.43 However, Lmo2 has no obligatory role in normal lymphoid development44 despite its presence in immature T cells.45,46,47 Rather, it is necessary early in hematopoiesis, since in mice without Lmo2, hematopoietic cells of any lineage fail to develop.38,48
Figure 3. Structure and Function of LIM Domains.
LMO2 is a member of the LMO (LIM-only) class of transcription factors first identified on the basis of its similarity to the related gene LMO1 — itself a target of the chromosomal translocation t(11;14)(p15;q11) in T-cell acute lymphoblastic leukemia.23,24 Proteins of the LIM-only family are almost entirely composed of zinc-binding finger-like motifs (Panel A) termed "LIM domains,"25,26,27 which function in protein–protein interactions.28,29,30 The intriguing aspect of the function of LMO2 is that this small molecule (156 amino acids ) has a key role in the development of blood and T-cell leukemogenesis. This role is partly explained by its structure, which comprises two LIM domains (Panel B),10 each of which is composed of two zinc-binding LIM fingers,31,32 in which a single zinc atom is shared by cysteine (Cys), histidine (His), or aspartate (Asp) residues. Although LMO2 is found in DNA-binding transcription-factor complexes involving the proteins TAL1 (or SCL), E47, LDB1, and GATA1 in normal erythroid cells (Panel A),33,34,35 a distinct complex of Lmo2, Tal1 (or Scl), E47, and Ldb1 was found in the leukemic T cells of Lmo2 transgenic mice.36,37 These findings imply that LMO2 may modulate gene expression by forming separate protein complexes at various normal sites of expression and in T-cell tumors. Panel B was adapted from Perez-Alvarado et al.32 with the permission of the publisher.
LMO2 as a Target of Retroviral Activation
The finding of retroviral insertion into LMO2 in two patients with X-linked SCID in whom leukemia developed8,9 and in a third patient who is free of leukemia at this time49 raises the crucial question whether the LMO2 gene is a preferred or even specific target in retrovirus-mediated gene therapy. Since LMO2 is only one of many genes activated by chromosomal translocations in T-cell ALL (Table 1), why was it apparently activated specifically in the X-linked SCID gene-therapy trial? Part of the answer lies in the mechanism of X-linked SCID, part in the gene-therapy procedure itself, and part in the biology of LMO2.
Table 1. Oncogenes Involved in Chromosomal Translocations in T-Cell Acute Leukemias.
Because X-linked SCID is characterized by a specific failure of T-cell production, whereas other types of hematopoietic cells are present in normal numbers (Figure 1B), the infusion of immature CD34+ hematopoietic cells into affected patients results in preferential engraftment of the T-cell lineage over other lineages. In the French trial, in which the numbers of T cells were restored to normal, all the new T cells contained the retroviral insertion, whereas the incidence of the integration of IL2Rc into cells of other lineages was typically 1 percent or less.2 This difference is due to two factors: progenitors in the graft of other lineages must compete with the normal numbers of resident progenitors, and progenitors without an IL2Rc insert can engraft and develop into lineages other than T cells, whereas only progenitors that express IL2Rc can form T cells.
This bias favoring T-cell production from engrafted stem cells would itself be expected to favor the onset of T-cell leukemia rather than other types of leukemia, if such an outcome were to occur. This is an important consideration in the context of the anticipated broad use of gene therapy. The goal of most gene-therapy regimens will not be to reconstitute a single hematopoietic lineage and, thus, with the benefit of hindsight, will not be expected to incur the same risk of leukemogenesis as it did in patients with X-linked SCID. However, the reconstitution of different hematopoietic lineages with the use of a retroviral vector or the direct use of retroviruses to target other somatic tissues could lead to distinct types of oncogenic events, each involving oncogenes peculiar to the type of cell that was targeted.
LMO2 is not specifically a lymphoid factor but rather is also expressed in early hematopoietic precursors, endothelial cells, and neural tissues.45,48,70 Reconstitution of the T-cell lineage in the patients with X-linked SCID was complete over a period of years, implying that it was derived from long-term repopulating stem cells. Retroviruses preferentially integrate into active genes and, indeed, near gene promoters.71 Thus, since LMO2 is expressed early in hematopoiesis, it is an ideal target for retroviral insertions in such cells. This property, combined with the capacity of LMO2 to transform T cells, made LMO2 an ideal candidate for retroviral oncogenesis in the X-linked SCID trial. However, since other T-cell oncogenes (e.g., TAL1 or SCL) have similar properties, it is likely that additional features of LMO2 render it susceptible to insertional mutagenesis.
There is also a numerical argument supporting the likelihood of the activation of LMO2. Given that there are as few as 30,000 genes in the human genome,72 that retroviral insertions favor expressed genes,71 that LMO2 is expressed in CD34+ cells, and that more than 90 million transduced CD34+ hematopoietic stem cells were reintroduced into the patients in the French trial, it is plausible that the reinfused cell population contained 10 to 100 progenitor cells with a retroviral insertion in LMO2, any one of which could undergo the additional mutations required to bring about T-cell leukemia.
Role of IL2Rc in Leukemogenesis
We also have to consider the possibility that the IL2Rc gene that was carried into hematopoietic precursors by the retrovirus collaborated in LMO2-mediated leukemogenesis. The IL2Rc subunit is a signaling subunit of the receptors for interleukin-2, 4, 7, 9, 15, and 21, all of which act as T-cell growth factors alone or in combination with other factors.73,74,75,76,77 The receptors for interleukin-2 and interleukin-15 have distinct subunits but share a subunit in addition to IL2Rc, whereas the receptors for interleukin-4, 7, 9, and 21 consist of distinct cytokine-binding subunits plus IL2Rc.7
Cytokine receptors with IL2Rc subunits have been implicated in leukemogenesis. For example, the interleukin-7 receptor, which is expressed in most cases of T-cell ALL, stimulates the synthesis of DNA and cell-cycle progression and inhibits apoptosis in the leukemic T cells.78,79,80 Moreover, adult T-cell leukemia cells express high-affinity interleukin-2 receptors, in part owing to the up-regulation of the interleukin-2 receptor subunit (IL-2R) by human T-cell lymphotropic virus I.81,82 If the overexpression of LMO2 blocks T-cell development in humans at a stage in which the chain of the receptor for interleukin-2 appears, as it does in mice,36,41 then such arrested cells in the patients with X-linked SCID would express two parts of the receptor for interleukin-2 (IL-2R and IL2Rc). These components may suffice to render the arrested T cells hypersensitive to signaling by the interleukin-2 growth factor. LMO2 may also have a direct role in the abrogation of growth-factor dependence; the target genes of the LMO2 complexes in leukemic T cells (Figure 3B) may include cytokines or their signaling components.
Chromosomal Translocation or Retroviral Activation plus Other Events
Much of what we know about the role of LMO2 in leukemia has been learned from the study of transgenic mice in which Lmo2-dependent leukemia develops.39,40,41,42,43 The long latency of leukemia in these mice implies that mutations are required in addition to the activation of Lmo2. The fact that the leukemias developed in the gene-therapy trial for X-linked SCID several years after engraftment suggests a similar course of events. The leukemia in one patient had an acquired mutation in another T-cell oncogene, TAL1, or SCL,9 and in the other patient, the onset of leukemia was immediately preceded by a varicella–zoster infection, suggesting that viral antigens were a stimulus for further leukemogenic mutations.83,84,85 However, antigenic stimulation is not a factor in Lmo2-mediated leukemogenesis in transgenic mice, because it can occur in the absence of antigen receptors on T cells.86
Conclusions
There are similarities in the causes of the T-cell leukemias that develop in Lmo2 transgenic mice, immunologically normal children with LMO2-associated chromosomal translocations, and patients with X-linked SCID who received gene therapy. These parallels are summarized in Figure 4.
Figure 4. Model of LMO2 Function in Leukemia.
T cells mature from common lymphocyte progenitors in the thymic cortex, where a series of differentiating cells expressing different combinations of the CD markers CD4, CD8, CD25, and CD44 occur. The most immature thymic T cells do not express CD4 and CD8 and are thus referred to as double-negative (DN) cells. The DN population goes through four stages of differentiation (from DN1 to DN4) cells, characterized by the expression of CD44 and CD25 markers as indicated. During this process, the recombination-activating gene recombinase genes (RAG1 and RAG2) are switched on in order to rearrange T-cell receptor (TCR) genes and eventually to allow the expression of T-cell receptors on the surface of CD4+, CD8+ (double-positive ) cells. These cells undergo positive and negative selection in the thymic cortex and medulla before they are released as mature, non–self-recognizing T cells expressing CD4 or CD8. The figure depicts this normal differentiation pathway and shows how the activation of LMO2 by chromosomal translocations (Panel A) or by the insertion of a retrovirus (Panel B) leads to leukemia. In Panel A, in transgenic mice with enforced expression of Lmo2, differentiation is blocked at the DN3 stage (RAG+), and overt leukemia eventually arises, presumably after secondary mutations in other genes. Sequence analysis of the LMO2-associated chromosomal translocations in humans shows that these mutations typically occur as a result of mistakes in RAG recombinase during rearrangements of T-cell receptors20,87 and thus most likely arise in the DN2 or DN3 cells in which this process occurs, as indicated. This process presumably causes a delay in T-cell differentiation similar to that seen in the Lmo2 transgenic mice. In Panel B, the effect of retroviral insertion into LMO2 in the patients with X-linked SCID who underwent gene therapy follows the differentiation of common lymphocyte precursors in the thymus by means of two mechanisms. First, the interleukin-2 receptor common subunit may increase proliferation of the lymphoid lineage and thus give rise to an accumulation of lymphoid precursor cells. Second, the enforced expression of LMO2 would cause a block in T-cell differentiation of the type observed in Lmo2 transgenic mice. The combined effects, together with secondary mutations, would give rise to the observed clonal T-cell leukemia.
T-cell development in the thymus can be divided into seven successive stages on the basis of the expression of surface markers on the maturing thymocytes (Figure 4). In transgenic mice with forced expression of Lmo2 in thymocytes, differentiation is blocked at the immature double-negative T-cell stage,37,44 before the appearance of overt leukemia (double-negative cells and double-positive cells are immature T cells in thymus that respectively lack or express CD8 and CD4). When leukemia eventually arises, it does not always have the double-negative phenotype, suggesting that further differentiation can occur before the final step of leukemic transformation. The long latency of leukemia in these mice suggests that mutations in other genes are needed to bring about leukemia. The break-point sequences of LMO2-associated chromosomal translocations in humans suggest aberrant involvement of the recombination-activating gene recombinase (the enzyme that directs the rearrangement of antigen-receptor genes) in the translocation process.20,87 Since recombination-activating gene recombinase is expressed from the first or second double-negative stage to the double-positive stage of development, the LMO2-associated chromosomal translocations in humans are likely to occur in these early double-negative cells. As in the mouse model, the accumulation of a pool of cells with blocked differentiation and susceptibility to further mutations brings about overt leukemia (Figure 4A).21
By analogy, developing T cells with a retroviral insertion in LMO2 in the patients with X-linked SCID should be blocked at the double-negative stage of T-cell differentiation and undergo the mutational changes required for the development of leukemia (Figure 4B). The proposed effects of retroviral insertion into LMO2 are twofold: forced expression of LMO2 to block T-cell differentiation, and expression of the IL2Rc chain to facilitate signals that induce cell division in the lymphoid lineage. The finding that an LMO2-associated retroviral insertion has been detected in a third patient without leukemia49 emphasizes that LMO2 was a common target in this gene-therapy trial. This trial reinforces the view that gene therapy has the potential to cure life-threatening diseases, a major development in medicine. In addition, the lessons learned from the trial will make gene therapy safer,88 given that some of the theoretical problems have become unfortunate realities, which can now be addressed by the redesign of vectors and approaches to transduction.
Source Information
From the Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Melbourne, Victoria, Australia (M.P.M.); and the Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom (T.H.R.).
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Related Letters:
LMO2 and Gene Therapy for Severe Combined Immunodeficiency
Fischer A., Abina S. H.-B., Thrasher A., von Kalle C., Cavazzana-Calvo M., Stone B. D., Rabbitts T. H., McCormack M. P.(Matthew P. McCormack, Ph.)
Figure 1. Normal (Panel A), Defective (Panel B), and Malignant (Panels D and E) Hematopoiesis in Persons with and without X-Linked Severe Combined Immunodeficiency (SCID), and Correction of the Lymphocyte Defect with the Use of Gene Therapy (Panel C).
In Panel A, normal hematopoiesis involves differentiation of pluripotential hematopoietic stem cells expressing the CD34 surface protein (CD34+) into two main committed progenitors: common myeloid and common lymphoid progenitors.3,4,5,6 Common myeloid progenitors can differentiate into all cells of the myeloerythroid lineage (granulocytes, macrophages, megakaryocytes, erythroid cells), and common lymphoid progenitors can differentiate into T cells, B cells, and natural killer cells. Boys with X-linked SCID are deficient in the common chain of the interleukin-2 receptor (IL2Rc), causing failure of normal lymphopoiesis (Panel B).7 Gene therapy was used to correct the defect in X-linked SCID (Panel C). CD34+ hematopoietic stem cells were infected ex vivo with a retrovirus encoding IL2Rc, and transduced autologous cells were reimplanted. Since the hematopoietic stem cells are progenitors of all hematopoietic cells, the transduced cells contribute cells carrying the integrated IL2Rc-retroviral DNA to all lineages, including the lymphocyte lineage, thereby correcting the immunodeficiency.2 The proportion of cells carrying the inserted retrovirus in the lymphoid lineages far outweighs that in other lineages. Unfortunately, T-cell leukemia developed in two of the patients successfully treated in this way (Panel D).8 In both children, the T-cell acute lymphoblastic leukemia (T-cell ALL) arose in cells in which the retroviral integration site was within or very close to the T-cell oncogene LMO2.9 This enforced LMO2 gene activation effectively mimics the molecular event that led to the discovery of the LMO2 gene — namely, chromosomal translocation in T-cell leukemia.10,11 In T-cell ALL involving the chromosomal translocation t(11;14)(p13;q11) or t(7;11)(q35;p13), activation of LMO2 occurs by means of the break point of these translocations, near a T-cell receptor gene, and its activation leads to leukemia (Panel E).
In a recent successful trial of gene therapy in Paris, CD34+ hematopoietic stem cells from patients with X-linked SCID were cultured ex vivo and transduced with a defective retroviral vector, based on the Moloney murine leukemia virus that carried the human IL2Rc gene.2 These transduced cells were reintroduced into the patients, who were then able to produce T cells and natural killer cells (Figure 1C). Levels of T cells in the blood reached normal values within a few months and have remained so for several years, although low numbers of natural killer cells persist.2 This result is the first clear demonstration of a clinical benefit of gene therapy and is a major step in the correction of inherited diseases, especially those involving deficiencies of hematopoietic cells.
A Major Adverse Effect of Viral Gene Therapy
Although gene therapy for X-linked SCID is a most important advance, it has sadly also uncovered a major adverse effect, which vividly demonstrates an intrinsic danger of retroviral vectors. Retroviral vectors are used in gene therapy because they can stably insert themselves into the genome of host cells. However, retroviruses of the type used in the French trial can also enhance the expression of cellular genes that surround the site of insertion of the vector through insertional mutagenesis.13 The possibility that the retroviruses used in gene therapy may cause insertional mutagenesis, thereby evoking untoward side effects, has been discussed for many years.14,15 Moreover, experiments in mice have shown that retroviral insertional mutagenesis causes leukemia and have yielded many candidate leukemia oncogenes.13,16,17,18 The risk of oncogenic insertional mutagenesis in humans as a consequence of gene therapy with the use of a retroviral vector was, however, deemed to be low. But the outcome of the X-linked SCID trial has shown that this risk is a major problem because, although 9 of 10 children treated with the IL2Rc-rescue protocol are in remission (long-term follow-up is essential before they can be considered to be cured), a T-cell acute leukemia-like syndrome has developed in 2 of the boys (Figure 1D).8,9 This event has been attributed to the integration of the retrovirus into a known T-cell oncogene, LMO2,9 in the deregulated T-cell clones (Figure 2), thereby contributing to the development of T-cell leukemia by causing aberrant expression of LMO2.
Figure 2. Molecular Architecture of the LMO2 Gene and Break Points for Retroviral Insertion or Chromosomal Translocations.
The LMO2 gene is located on the short arm (p) of human chromosome 11 at band 13 (11p13). The gene has two transcriptional promoters19 and comprises six exons, of which exons 4, 5, and 6 code for protein. Chromosomal translocations involving LMO2 are found in T-cell acute leukemias, and the chromosome 11p13 break point can be paired with either the T-cell receptor gene (TCR) on chromosome 14 (band q11) or the T-cell receptor gene (TCR) on chromosome 7 (band q35).20 All known chromosomal translocations occur upstream of one or both of the LMO2 promoters.21 The same feature is true of the X-linked severe combined immunodeficiency retroviral (MFGc) insertion sites associated with leukemia, which were found either just ahead of LMO2 exon 1 or between exons 1 and 2.9 In both circumstances the LMO2 protein product is identical before and after the genetic abnormality has occurred, and it is the enforced expression of LMO2 that influences T-cell development and leukemia. IL2Rc denotes interleukin-2 receptor common subunit, Met methionine protein translation-initiation codon, and stop protein-translation stop codon.
LMO2, a Quintessential Chromosomal Translocation Oncogene
The occurrence of T-cell leukemia after forced expression of the LMO2 gene by a retrovirus is not surprising, given that LMO2 is involved in spontaneous cases of T-cell leukemia.10,11 LMO2 is found at the break points of the t(11;14)(p13;q11) or the t(7;11)(q35;p13) translocations in T-cell acute lymphoblastic leukemia (T-cell ALL)20,22 (Figure 2) and encodes a LIM-only protein (LMO2), which acts as a bridging molecule in transcription-factor complexes (Figure 3). LMO2 is present in the progenitors of all hematopoietic lineages before its expression is down-regulated in all except the erythroid lineage.38 In T cells, down-regulation of the protein appears to be crucial, since T-cell leukemia develops in mice that constitutively express Lmo2 in the thymus.39,40 The overt leukemia is preceded by an accumulation of immature thymic T cells, indicating that deregulated Lmo2 increases susceptibility to leukemia by blocking the differentiation of T cells.36,41,42 Only T-cell leukemia develops in transgenic mice, even though Lmo2 is expressed in many tissues, implying that this gene is specifically a T-cell oncogene.43 However, Lmo2 has no obligatory role in normal lymphoid development44 despite its presence in immature T cells.45,46,47 Rather, it is necessary early in hematopoiesis, since in mice without Lmo2, hematopoietic cells of any lineage fail to develop.38,48
Figure 3. Structure and Function of LIM Domains.
LMO2 is a member of the LMO (LIM-only) class of transcription factors first identified on the basis of its similarity to the related gene LMO1 — itself a target of the chromosomal translocation t(11;14)(p15;q11) in T-cell acute lymphoblastic leukemia.23,24 Proteins of the LIM-only family are almost entirely composed of zinc-binding finger-like motifs (Panel A) termed "LIM domains,"25,26,27 which function in protein–protein interactions.28,29,30 The intriguing aspect of the function of LMO2 is that this small molecule (156 amino acids ) has a key role in the development of blood and T-cell leukemogenesis. This role is partly explained by its structure, which comprises two LIM domains (Panel B),10 each of which is composed of two zinc-binding LIM fingers,31,32 in which a single zinc atom is shared by cysteine (Cys), histidine (His), or aspartate (Asp) residues. Although LMO2 is found in DNA-binding transcription-factor complexes involving the proteins TAL1 (or SCL), E47, LDB1, and GATA1 in normal erythroid cells (Panel A),33,34,35 a distinct complex of Lmo2, Tal1 (or Scl), E47, and Ldb1 was found in the leukemic T cells of Lmo2 transgenic mice.36,37 These findings imply that LMO2 may modulate gene expression by forming separate protein complexes at various normal sites of expression and in T-cell tumors. Panel B was adapted from Perez-Alvarado et al.32 with the permission of the publisher.
LMO2 as a Target of Retroviral Activation
The finding of retroviral insertion into LMO2 in two patients with X-linked SCID in whom leukemia developed8,9 and in a third patient who is free of leukemia at this time49 raises the crucial question whether the LMO2 gene is a preferred or even specific target in retrovirus-mediated gene therapy. Since LMO2 is only one of many genes activated by chromosomal translocations in T-cell ALL (Table 1), why was it apparently activated specifically in the X-linked SCID gene-therapy trial? Part of the answer lies in the mechanism of X-linked SCID, part in the gene-therapy procedure itself, and part in the biology of LMO2.
Table 1. Oncogenes Involved in Chromosomal Translocations in T-Cell Acute Leukemias.
Because X-linked SCID is characterized by a specific failure of T-cell production, whereas other types of hematopoietic cells are present in normal numbers (Figure 1B), the infusion of immature CD34+ hematopoietic cells into affected patients results in preferential engraftment of the T-cell lineage over other lineages. In the French trial, in which the numbers of T cells were restored to normal, all the new T cells contained the retroviral insertion, whereas the incidence of the integration of IL2Rc into cells of other lineages was typically 1 percent or less.2 This difference is due to two factors: progenitors in the graft of other lineages must compete with the normal numbers of resident progenitors, and progenitors without an IL2Rc insert can engraft and develop into lineages other than T cells, whereas only progenitors that express IL2Rc can form T cells.
This bias favoring T-cell production from engrafted stem cells would itself be expected to favor the onset of T-cell leukemia rather than other types of leukemia, if such an outcome were to occur. This is an important consideration in the context of the anticipated broad use of gene therapy. The goal of most gene-therapy regimens will not be to reconstitute a single hematopoietic lineage and, thus, with the benefit of hindsight, will not be expected to incur the same risk of leukemogenesis as it did in patients with X-linked SCID. However, the reconstitution of different hematopoietic lineages with the use of a retroviral vector or the direct use of retroviruses to target other somatic tissues could lead to distinct types of oncogenic events, each involving oncogenes peculiar to the type of cell that was targeted.
LMO2 is not specifically a lymphoid factor but rather is also expressed in early hematopoietic precursors, endothelial cells, and neural tissues.45,48,70 Reconstitution of the T-cell lineage in the patients with X-linked SCID was complete over a period of years, implying that it was derived from long-term repopulating stem cells. Retroviruses preferentially integrate into active genes and, indeed, near gene promoters.71 Thus, since LMO2 is expressed early in hematopoiesis, it is an ideal target for retroviral insertions in such cells. This property, combined with the capacity of LMO2 to transform T cells, made LMO2 an ideal candidate for retroviral oncogenesis in the X-linked SCID trial. However, since other T-cell oncogenes (e.g., TAL1 or SCL) have similar properties, it is likely that additional features of LMO2 render it susceptible to insertional mutagenesis.
There is also a numerical argument supporting the likelihood of the activation of LMO2. Given that there are as few as 30,000 genes in the human genome,72 that retroviral insertions favor expressed genes,71 that LMO2 is expressed in CD34+ cells, and that more than 90 million transduced CD34+ hematopoietic stem cells were reintroduced into the patients in the French trial, it is plausible that the reinfused cell population contained 10 to 100 progenitor cells with a retroviral insertion in LMO2, any one of which could undergo the additional mutations required to bring about T-cell leukemia.
Role of IL2Rc in Leukemogenesis
We also have to consider the possibility that the IL2Rc gene that was carried into hematopoietic precursors by the retrovirus collaborated in LMO2-mediated leukemogenesis. The IL2Rc subunit is a signaling subunit of the receptors for interleukin-2, 4, 7, 9, 15, and 21, all of which act as T-cell growth factors alone or in combination with other factors.73,74,75,76,77 The receptors for interleukin-2 and interleukin-15 have distinct subunits but share a subunit in addition to IL2Rc, whereas the receptors for interleukin-4, 7, 9, and 21 consist of distinct cytokine-binding subunits plus IL2Rc.7
Cytokine receptors with IL2Rc subunits have been implicated in leukemogenesis. For example, the interleukin-7 receptor, which is expressed in most cases of T-cell ALL, stimulates the synthesis of DNA and cell-cycle progression and inhibits apoptosis in the leukemic T cells.78,79,80 Moreover, adult T-cell leukemia cells express high-affinity interleukin-2 receptors, in part owing to the up-regulation of the interleukin-2 receptor subunit (IL-2R) by human T-cell lymphotropic virus I.81,82 If the overexpression of LMO2 blocks T-cell development in humans at a stage in which the chain of the receptor for interleukin-2 appears, as it does in mice,36,41 then such arrested cells in the patients with X-linked SCID would express two parts of the receptor for interleukin-2 (IL-2R and IL2Rc). These components may suffice to render the arrested T cells hypersensitive to signaling by the interleukin-2 growth factor. LMO2 may also have a direct role in the abrogation of growth-factor dependence; the target genes of the LMO2 complexes in leukemic T cells (Figure 3B) may include cytokines or their signaling components.
Chromosomal Translocation or Retroviral Activation plus Other Events
Much of what we know about the role of LMO2 in leukemia has been learned from the study of transgenic mice in which Lmo2-dependent leukemia develops.39,40,41,42,43 The long latency of leukemia in these mice implies that mutations are required in addition to the activation of Lmo2. The fact that the leukemias developed in the gene-therapy trial for X-linked SCID several years after engraftment suggests a similar course of events. The leukemia in one patient had an acquired mutation in another T-cell oncogene, TAL1, or SCL,9 and in the other patient, the onset of leukemia was immediately preceded by a varicella–zoster infection, suggesting that viral antigens were a stimulus for further leukemogenic mutations.83,84,85 However, antigenic stimulation is not a factor in Lmo2-mediated leukemogenesis in transgenic mice, because it can occur in the absence of antigen receptors on T cells.86
Conclusions
There are similarities in the causes of the T-cell leukemias that develop in Lmo2 transgenic mice, immunologically normal children with LMO2-associated chromosomal translocations, and patients with X-linked SCID who received gene therapy. These parallels are summarized in Figure 4.
Figure 4. Model of LMO2 Function in Leukemia.
T cells mature from common lymphocyte progenitors in the thymic cortex, where a series of differentiating cells expressing different combinations of the CD markers CD4, CD8, CD25, and CD44 occur. The most immature thymic T cells do not express CD4 and CD8 and are thus referred to as double-negative (DN) cells. The DN population goes through four stages of differentiation (from DN1 to DN4) cells, characterized by the expression of CD44 and CD25 markers as indicated. During this process, the recombination-activating gene recombinase genes (RAG1 and RAG2) are switched on in order to rearrange T-cell receptor (TCR) genes and eventually to allow the expression of T-cell receptors on the surface of CD4+, CD8+ (double-positive ) cells. These cells undergo positive and negative selection in the thymic cortex and medulla before they are released as mature, non–self-recognizing T cells expressing CD4 or CD8. The figure depicts this normal differentiation pathway and shows how the activation of LMO2 by chromosomal translocations (Panel A) or by the insertion of a retrovirus (Panel B) leads to leukemia. In Panel A, in transgenic mice with enforced expression of Lmo2, differentiation is blocked at the DN3 stage (RAG+), and overt leukemia eventually arises, presumably after secondary mutations in other genes. Sequence analysis of the LMO2-associated chromosomal translocations in humans shows that these mutations typically occur as a result of mistakes in RAG recombinase during rearrangements of T-cell receptors20,87 and thus most likely arise in the DN2 or DN3 cells in which this process occurs, as indicated. This process presumably causes a delay in T-cell differentiation similar to that seen in the Lmo2 transgenic mice. In Panel B, the effect of retroviral insertion into LMO2 in the patients with X-linked SCID who underwent gene therapy follows the differentiation of common lymphocyte precursors in the thymus by means of two mechanisms. First, the interleukin-2 receptor common subunit may increase proliferation of the lymphoid lineage and thus give rise to an accumulation of lymphoid precursor cells. Second, the enforced expression of LMO2 would cause a block in T-cell differentiation of the type observed in Lmo2 transgenic mice. The combined effects, together with secondary mutations, would give rise to the observed clonal T-cell leukemia.
T-cell development in the thymus can be divided into seven successive stages on the basis of the expression of surface markers on the maturing thymocytes (Figure 4). In transgenic mice with forced expression of Lmo2 in thymocytes, differentiation is blocked at the immature double-negative T-cell stage,37,44 before the appearance of overt leukemia (double-negative cells and double-positive cells are immature T cells in thymus that respectively lack or express CD8 and CD4). When leukemia eventually arises, it does not always have the double-negative phenotype, suggesting that further differentiation can occur before the final step of leukemic transformation. The long latency of leukemia in these mice suggests that mutations in other genes are needed to bring about leukemia. The break-point sequences of LMO2-associated chromosomal translocations in humans suggest aberrant involvement of the recombination-activating gene recombinase (the enzyme that directs the rearrangement of antigen-receptor genes) in the translocation process.20,87 Since recombination-activating gene recombinase is expressed from the first or second double-negative stage to the double-positive stage of development, the LMO2-associated chromosomal translocations in humans are likely to occur in these early double-negative cells. As in the mouse model, the accumulation of a pool of cells with blocked differentiation and susceptibility to further mutations brings about overt leukemia (Figure 4A).21
By analogy, developing T cells with a retroviral insertion in LMO2 in the patients with X-linked SCID should be blocked at the double-negative stage of T-cell differentiation and undergo the mutational changes required for the development of leukemia (Figure 4B). The proposed effects of retroviral insertion into LMO2 are twofold: forced expression of LMO2 to block T-cell differentiation, and expression of the IL2Rc chain to facilitate signals that induce cell division in the lymphoid lineage. The finding that an LMO2-associated retroviral insertion has been detected in a third patient without leukemia49 emphasizes that LMO2 was a common target in this gene-therapy trial. This trial reinforces the view that gene therapy has the potential to cure life-threatening diseases, a major development in medicine. In addition, the lessons learned from the trial will make gene therapy safer,88 given that some of the theoretical problems have become unfortunate realities, which can now be addressed by the redesign of vectors and approaches to transduction.
Source Information
From the Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Melbourne, Victoria, Australia (M.P.M.); and the Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom (T.H.R.).
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LMO2 and Gene Therapy for Severe Combined Immunodeficiency
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