Molecular Basis of Mature T-Cell Leukemia

Mature T-cell leukemias are characterized by the accumulation of mature lymphocytes in the peripheral blood. The disease is usually seen in patients older than 50 years and is rare before the fourth decade of life. It is more common in males than in females. These T-cell leukemias represent a heterogeneous group of diseases resulting from the clonal proliferation of post-thymic immunocompetent lymphoid cells.^{1 - 2} The further classification of these leukemias is still an unresolved issue, especially as it is not clear whether T-cell chronic lymphocytic leukemia (T-CLL) and T-cell prolymphocytic leukemia (T-PLL) are separate entities.^{1 - 2} T-cell prolymphocytic leukemia is the common form of chronic T-cell leukemia, showing characteristics of prolymphocytic cells with T-cell phenotype. However, smaller, more mature-appearing lymphocytes, resembling B-cell chronic lymphocytic leukemia, but expressing T-cell markers such as CD5 and T-cell receptor rearrangement, may be a separate form of leukemia, called T-CLL.^{1 - 2} Patients with T-PLL or T-CLL typically have significantly elevated white blood cell counts (in excess of 200 × 10³/µL), often accompanied by splenomegaly and/or hepatomegaly. Anemia and thrombocytopenia are detected in less than half of the patients. Primary nodal presentation is unusual, whereas mucosal and cutaneous manifestations are frequent.^{3 - 4} Pleural effusions and ascites are also commonly seen features. The disease progresses in a moderately aggressive way (median survival time, 7 months) and is generally not curable.^{3 - 4} This leukemic disorder occurs with high frequency in patients with ataxia-telangiectasia (AT), an autosomalrecessive disorder characterized by predisposition to malignancies, among other manifestations, and caused by mutations in the ATM (mutated in AT) gene.⁵

Adult T-cell leukemia or adult T-cell leukemia/lymphoma (ATL) is a unique clinicopathological disorder caused by the human T-cell lymphotropic virus, a retrovirus.² It shares with T-PLL/T-CLL the mature T-cell phenotype and the clinical features such as splenomegaly and hepatomegaly, but it mostly occurs in an acute form.⁵ The most common cytogenetic features of T-PLL/T-CLL and ATL are clonal rearrangements at 14q32.1, including inversion inv(14)(q11;q32) and translocation t(14;14)(q11;q32).^{6 - 8} In addition, rearrangements of the TCL1 (T-cell leukemia/lyphoma 1) locus have been observed with T-cell clonal expansions occurring in patients with AT before the onset of frank leukemia.⁹ Studies of other translocations associated with human leukemias and lymphomas uncovering the existence and/or significance of important oncogenes including c-myc, bcl2, abl, and ALL1^{10 - 15} had prompted the investigation of the rearrangements of 14q32.1.

We used a MEDLINE search to identify all published English-language articles (we are not aware of literature in other languages relevant to this topic) on T-cell leukemias and related topics from 1980 through 2001. From more than 7000 citations, 205 articles were identified as relevant to the topic of molecular mechanisms of T-cell leukemias. Most of these articles were focused on immature T-cell leukemias and/or the role of the T-cell receptor genes. Ninety-six articles were relevant to the topic of mature T-cell leukemias and were carefully examined. After elimination of redundant articles and articles that contributed only peripheral data relevant to the topic in question, 55 articles remained and were included in this study.

In this article, we first review and explain the chromosomal rearrangement events leading to the transformation of T cells and the development of leukemia. We then summarize data describing the genes involved in these rearrangements, ie, the genes of the TCL1locus, presenting an overview of the genomic structure of this locus and the expression analysis of the genes in question. Finally, we review recent publications describing the biochemical pathways involving the TCL1 gene and present our current view of the chain of molecular events connecting the activation of the TCL1 locus to the transformation of T cells.

As mentioned, rearrangements at 14q32.1 are associated with mature T-cell leukemias such as T-PLL/T-CLL and ATL^{16 - 17} and with chronic T-cell leukemias that develop in patients with the immunodeficiency syndrome AT.⁵ Thus, the locus involved in these abnormalities is called the TCL1 (T-cell leukemia/lymphoma 1) locus. Typically, the TCL1 locus rearranges with the TCR (T-cell receptor) α/δ locus at chromosome 14q11 (Figure 1),^{18 - 19} but it can also rearrange with the TCRβ locus at chromosome 7q35.¹⁹ Because the TCL1 locus and the TCR α/δ locus (TCRα locus only shown in Figure 1) reside on the same chromosome, these rearrangements can either be inversions inv(14)(q11;q32) or reciprocal translocations t(14;14)(q11;q32).^{19 - 20} On the chromosome 14 carrying the inversion inv(14)(q11;q32), the rearranged TCL1 locus is linked to a J/D segment of the TCRα/δ receptor (the small D segment is located between the V segment and the J segment). The abnormal chromosome 14 showing the translocation t(14;14)(q11;q32) contains 2 copies of the TCL1 locus: 1 rearranged TCL1 copy juxtaposed with the J/D segment of the TCRα/δ receptor and 1 normal copy located in the distal part of the chromosome; in this case, the orientation of the TCRα enhancer (functions in multiplying gene activation) is opposite to that of the inversion inv(14)(q11;q32) (Figure 1). Since the expression of the genes at the TCL1 locus is induced by both types of rearrangements,²⁰ the orientation of the TCRα enhancer respective to the TCL1 locus seems of no consequence.

The frequent involvement of rearrangements of the TCL1 locus in T-cell leukemias suggested the existence of an oncogene in this region important for the initiation and/or progression of T-cell leukemias.⁸ The first step in the isolation of the gene in question involved the construction of a P1 contig covering 14q32.1 and mapping several 14q32.1 breakpoints within this region.¹⁹ Genomic analysis of this contig revealed that an approximately 120-kilobase (kb) DNA segment is located between the 2 breakpoint clusters reported in various T-PLL/T-CLL samples and cell lines (Figure 2). Transcriptional mapping of this 120-kb region led to the discovery of the TCL1 gene.²⁰ The gene consists of 4 exons with an open reading frame of 114 amino acids and a 3′ untranslated region of about 800 nucleotides (Figure 2).²⁰ Comparison with other known proteins revealed that the Tcl1 protein shows 41% amino acid sequence identity with Mtcp1 protein.²¹ Interestingly, the MTCP1 gene, also a member of the TCL1 gene family, located at Xq28, is involved in rare cases of mature T-cell leukemia with a t(X;14)(q28;q11) translocation.^{21 - 24} Subsequent studies of the TCL1 gene have shown its involvement in the TCL1 locus rearrangements and that its activation (initiation of expression) is the causal event in the pathogenesis of T-cell malignancies.^{20 ,25} Although 14q32.1 rearrangements typically lead to TCL1 activation, some reported cases of T-cell malignancies showing rearrangement at 14q32.1 did not show TCL1 expression.^{26 - 28} This suggested that additional oncogene(s) may be located in the region.²⁸

Recent genomic analysis of the 14q32.1 breakpoint region revealed 3 additional genes: TNG1 (TCL1 neighboring gene 1), TNG2, and TCL1b (Figure 2).^{29 - 32} The TNG1 and TNG2 genes encode proteins with no significant homology to known proteins or protein motifs.^{29 ,32} The TCL1b gene encodes a 14-kd protein of 128 amino acids. Interestingly, Tcl1b protein shows 60% similarity to the Tcl1 and Mtcp1 proteins, although the TCL1b gene contains a small insertion when compared with TCL1.^{30 - 31} Of note, TCL1 is located only 15 kb telomeric of TCL1b in the opposite orientation of transcription (Figure 2).

In developing mouse models for T-cell leukemia, we recently reported the genomic structure of the mouse Tcl1 locus located on chromosome 12. The combination of sequencing a bacterial artificial chromosome clone containing this locus and expressed sequence tag analysis of murine Tcl1 and Tcl1b homologs revealed that the murine Tcl1 locus contains 5 different Tcl1b genes (Tcl1b1-Tcl1b5) adjacent to the Tcl1 gene (Figure 2).²⁹ Interestingly, all 6 genes—encoding similar but distinct proteins—show mRNA expression and span only an 80-kb DNA segment on the murine chromosome 12 (Figure 2). Although the members of the TCL1 and Tcl1 gene families show only 30% to 40% homology, all of them contain a conserved hydrophobic core.²⁹

Since its discovery, the expression of TCL1 in normal and tumor tissues has been studied in great detail. Nonlymphatic tissues or cell lines tested are negative for TCL1 expression, with the exception of normal ovarian tissue and ovarian tumor tissue.²⁰ Most cells of B-cell lineage express TCL1, including pre B-cells, surface IgM-expressing virgin B cells, mantle cells, and, to a lesser extent, germinal center B cells, although it is not expressed in postgerminal center B cells, ie, plasma cells.^{28 ,33} However, T cells express TCL1 at an early stage, with double-negative markers (CD4⁻CD8⁻),^{20 ,33} whereas more mature T cells do not normally express TCL1. Expression analysis of TCL1 in T-PLL/T-CLL samples revealed the activation of the gene in almost 100% of cases studied.^{20 ,33 - 34} Some of the breakpoints that are seen in these types of leukemias are shown in Figure 2.^{19 - 20} In addition to spontaneous cases, T-PLL/T-CLL is frequently seen in patients with AT.^{9 ,16 ,18} These patients have a high level of specific chromosome translocations that can be easily observed in peripheral T cells and are associated with a greatly increased risk of leukemia/lymphoma, mainly of T-cell origin.¹⁶ Approximately 10% of all AT homozygotes develop a malignancy, and 70-fold and 250-fold increased risks for leukemias and lymphomas, respectively, have been reported.¹⁶ The specific chromosome translocations in these patients with AT involving chromosome 14q32.1 lead to the expression of TCL1.^{9 ,20 ,35} Expression of TCL1 was also reported in the preleukemic clone cells of patients with AT containing the primary translocation alone.⁹ Some expression of TCL1 can also be detected in randomly selected patients with AT without large cytogenetic clones and without any evidence of leukemic change.³⁶ Consistent with these data, the leukemic T-cell line SupT 11, which carries a t(14:14)(q11;q32) chromosomal translocation, also shows activation of TCL1.²⁰ The activation of TCL1 in T-PLL/T-CLL is well-documented, whereas the expression of this gene in ATLs is still controversial. Narducci et al¹⁷ reported the expression of TCL1 in 10 of 10 ATL specimens, but in 2 other studies,^{27 - 28} expression of this gene was not detected.

Although TCL1 functions as an oncogene predominantly in T cells, a recent report demonstrated its involvement in acquired immunodeficiency syndrome (AIDS)–related B-cell lymphomas, particularly in immunoblastic lymphoma plasmacytoid (IBLP).³⁷ AIDS IBLP is not associated with any predominant genetic alteration and is thought to derive from postgerminal center B cells.³⁷ Although B cells at this stage usually do not contain it, Tcl1 protein was found in the majority of AIDS IBLP tumors examined.^{37 - 38} Since rearrangements of 14q32.1 have not been reported in B-cell neoplasms, activation of TCL1 in B-cell lymphomas likely does not occur via the same chromosomal alterations involved with T-PLL/T-CLL. However, TCL1 can also be activated by hypomethylation of its promoter.³⁹ Determination of whether this mechanism also occurs in the case of AIDS IBLP requires investigation.

The expression pattern of the other 3 genes in the TCL1 locus, TCL1b, TNG1, and TNG2, is similar to that of TCL1: they are not expressed in most normal T cells, but activated in the SupT11 cell line and in some samples from patients with T-PLL.^{29 - 30} Interestingly, the genes of the murine Tcl1 locus show a different expression pattern. These genes are expressed only at the earliest stages of mouse embryogenesis and in oocytes.²⁹ Murine Tcl1 and Tcl1b genes were not expressed in any of the adult mouse tissues analyzed, including lymphoid tissues,²⁹ although a detailed study of expression during mouse hematopoiesis is yet to be carried out.

To investigate whether TCL1 is an oncogene involved in tumor initiation, human TCL1 cDNA was introduced into fertilized mouse eggs under the control of the lck proximal promoter.²⁵ Around the age of 6 months, the resulting transgenic mice developed proliferations of premalignant T cells containing Tcl1 protein. At the average age of 15 months, these transgenic animals had mature T-cell leukemias similar to human T-PLL/T-CLL, although unlike in humans, in whom the T-PLL/T-CLLs consist mostly of CD4⁺CD8⁻T cells, these leukemias consisted predominantly of CD4⁻CD8⁺ T cells.²⁵ The development of these leukemias proved that deregulation (caused by rearrangement) of the TCL1 oncogene initiates the process of leukemogenesis. Interestingly, transgenic mice expressing MTCP1, under the control of the CD2 promoter (another T-cell specific promoter), developed a similar type of leukemia, also having predominantly a CD4⁻CD8⁺ phenotype.⁴⁰ Therefore, deregulation of both of these members of the TCL1 gene family in T cells resulted in the development of T-cell leukemias in transgenic mice at older ages. These leukemias had a phenotype similar to that of mature T-cell leukemias in humans.²⁵ Since both TCL1 and TCL1b are activated in T-cell leukemias,^{20 ,30} the investigation of TCL1b transgenic mice and TCL1/TCL1b double-transgenic mice will be of considerable interest, specifically with respect to the age of onset of leukemia.

The activation of TCL1 as the causal event in the pathogenesis of mature T-cell leukemia is well documented.^{20 ,25} Nevertheless, the exact molecular mechanism of Tcl1 oncogenic action was not known until recently. To study the molecular role of Tcl1 in T-cell leukemia, several pathways involved in T-cell apoptosis and/or proliferation were considered for further investigation. One of the pathways having a significant role in signal transduction in T cells is the PI3-kinase–dependent Akt/protein kinase B oncogenic pathway. Akt is the human homolog of v-akt, a gene isolated from the retrovirus AKT8, which causes T-cell lymphoma in mice.⁴¹ The Akt kinase is an important signaling molecule in the transduction of proliferative and antiapoptotic signals in T cells.⁴² The main function of Akt lies in the phosphorylation and thus regulation of a variety of proteins important for cell proliferation and survival including Raf, IKKα, and Bad.^{43 - 45} The Akt protein consists of 2 domains: a kinase domain and a pleckstrin homology domain that mediates protein-protein interactions. Insulin and various other growth and survival factors can activate the Akt kinase.⁴²

To investigate a possible connection between Tcl1 and Akt functions, the proteins were first tested for physical interaction by coimmunoprecipitation studies. These experiments revealed that Akt1(one of the 3 types of kinases) forms immune complexes with Tcl1⁴⁶ (Figure 3) and Mtcp1.⁴⁷ Furthermore, Tcl1 enhances the kinase activity of Akt,^{46 - 47} possibly by promoting the oligomerization of Akt⁴⁷ or by changing its conformation.⁴⁶

Akt is localized mostly in the cytoplasm of cells,⁴² whereas Tcl1 is located in both cytoplasm and the nucleus.⁴⁶ However, when Akt1 and Tcl1 occur in the same cells, both proteins are colocalized in the nucleus and cytoplasm, as shown by 2-color immunofluorescence.⁴⁶ Thus, Tcl1 interacts with Akt, enhances Akt kinase activity, and appears to promote the nuclear translocation of Akt.^{46 - 47} The oncogenic function of Akt involves the phosphorylation and regulation of critical proapoptotic and/or pro-survival factors such as Raf, Bad, and IKKα.^{43 - 45} Therefore, Tcl1 may play an oncogenic role by increasing the kinase activity of Akt.⁴⁶ Since all reported targets of Akt are cytoplasmic,^{43 - 45} the translocation of Akt to the nucleus via Tcl1 suggests the existence of nuclear targets for Akt important for T-cell development and neoplastic transformation.

Recently, we and others have identified 1 such target.^{48 - 49} The orphan nuclear receptor Nur77 (also known as NGFI-B or TR3) is a critical molecule for T-cell receptor–mediated apoptosis in immature thymocytes undergoing selection.⁵⁰ Nur77 is a transcription factor and its activity is mediated through a unique response element.^{51 - 52} Two recent reports demonstrated that Akt interacts with and phosphorylates Nur77 at Ser350 (located in the DNA binding domain of Nur77) in a PI3-kinase–dependent manner and thus inhibits the function of Nur77 as a proapoptotic transcription factor (Figure 3).^{48 - 49} Since Tcl1 increases Akt kinase activity, it seems likely that Tcl1 may enhance this inactivation of Nur77 and therefore promote cell survival (Figure 3),^{48 - 49} although additional investigations are necessary to define this role of Tcl1.

The rearrangements of 14q32.1 are the most common cytogenetic features of mature T-cell leukemia.⁸ The studies of chromosomal translocations and inversions at 14q32.1 in patients with T-PLL/T-CLL resulted in the identification of a TCL1 gene family that functions in the development, proliferation, and apoptosis of lymphoid cells.^{20 ,30}

Activation of TCL1 (and possibly TCL1b) appears to be a causal event in T-cell leukemia development, as was determined by studying the transgenic mouse TCL1 model,²⁵ although other genetic changes are probably required for the pathogenesis of this disease, since the mice develop a mature T-cell leukemia later in life.²⁵

The TCL1b, TNG1, and TNG2 genes are also activated by chromosomal rearrangement involving 14q32 and may be acting as oncogenes on their own or in combination with TCL1 or each other.^{29 - 30} Furthermore, the activation of TCL1 in leukemia is not limited to T cells: it is also activated and possibly plays a role in the pathogenesis of IBLP B-cell lymphoma in patients with AIDS.³⁷

The exact biological function of Tcl1 and members of its family is still not clearly defined, although recent studies demonstrated its involvement in the PI3-kinase–dependent Akt oncogenic pathway, which possibly includes the inactivation of the proapoptotic factor Nur77.^{46 - 49}

There is no cure for T-CLL/T-PLL. However, the outcome of patients with this disease has improved in the last few years due to the development of new drugs such as pentostatin⁵³ and the humanized monoclonal Campath-1H antibody.⁵³ Still, these drugs can prolong the survival rates of the patients to an average of 12 to 24 months only.^{53 - 54} To effectively treat T-CLL/T-PLL, it would be helpful to identify agents that interfere directly with the molecular pathways leading to this disease (ie, the oncogenic pathways involving the TCL1 gene family). This approach was recently found to be effective in the treatment of chronic myeloid leukemia (CML).⁵⁵ The BCR-ABL tyrosine kinase is known to be constitutively active in all CML cells.⁵⁵ STI571 (formerly known as CGP 57148B) is a drug that specifically inhibits the activity of this kinase and has had significant antileukemic activity in patients with CML in whom other treatments had failed.⁵⁵ These results demonstrate the essential role of BCR-ABL tyrosine kinase activity in CML and show the potential for development of drugs based on a specific molecular abnormality present in human malignancies.

The full understanding of the oncogenic pathway(s) used by the TCL1 oncogene and members of its family may provide important information useful in uncovering the molecular basis of leukemogenesis of T cells and may result in the development of novel targeted approaches to the treatment of this disease.