The Role of TAL1 in Hematopoiesis and Leukemogenesis

INTRODUCTION

Hematopoiesis comprises a series of steps, including the formation of earlyhematopoietic progenitor cells from mesoderm, the formation of hematopoieticstem cells (HSC), and their further differentiation into mature blood cells.Dysregulation of these processes in hematopoietic precursor cells often leadsto their abnormal differentiation and proliferation and, as a result, malignanttransformation. The transcription factor TAL1 is one of the main regulators ofhematopoiesis. It comprises a helix-loop-helix domain which binds to DNAthrough its regulatory regions, interacting with the E-box sequence (CANNTG,where N is any nucleotide), and GATA, Ets, and Runx factor binding sites[1]. It has been shown that inhibitionof TAL1 gene expression leads to a complete absence ofhematopoiesis in the yolk sac [2].In an adult organism, a mximum level of TAL1 expressionis characteristic of pluripotent HSCs, multipotent myeloid and lymphoidprogenitors, as well as erythroid and megakaryocytic cells[3]. TAL1 participates in the formationof complexes with various transcription factors (E47/E2A, LMO2,GATA1–3, Ldb1/2, Ldb1, ETO₂, Runx1, ERG, FLI1)[4, 5].The composition of the complex may vary. The composition of the complexdetermines the intracellular targets it interacts with, activating orinhibiting the expression of the factors associated with differentiation of myeloid and lymphoid cells[6-8].An abnormal expression level or mutations in genes whose translation products comprisethe SCL complex can lead to malignant transformation of blood cells. Approximately 60%of cases of T-cell acute lymphoblastic leukemia (T-ALL) are characterized by an abnormally highlevel of TAL1 expression [9].Mutant forms of TAL1 in lymphoid and myeloid leukemia cellsare diagnosed in 20% of patients [10].The promoter portion of the C-KIT gene encoding the receptortyrosine kinase is considered as one of the main TAL1 targets in malignantblood cells. In some cases, it has been shown that progression of malignanthematological diseases (including acute myeloid leukemia) is accompanied by anabnormally high expression of C-KIT[11, 12].

TAL1: GENE STRUCTURE, KNOWN ISOFORMS OF THE PROTEIN AND THEIR FUNCTION IN HEMATOPOIESIS

The TAL1 gene locus is located on human chromosome 1. TAL1belongs to the family of transcription factors that possess a helix-loop-helix(bHLH) motif. The TAL1 gene contains six exons, including thecoding exons 4–6. According to the PubMed database as of 2017, sixdifferent transcripts of the TAL1 gene have been described(Fig. 1).There are two isoforms to the TAL1 protein: a long(TAL1-l) one, with a molecular weight of 34.3 kDa and composed of 331 aminoacid residues, and a short (TAL1-s) one, consisting of 156 amino acid residues.The TAL1-l to TAL1-s ratio differs in megacaryocyte-erythroid cells[13]. TAL1 pre-mRNA isalternatively spliced, producing mRNA without the exons 1–4. TheETO₂-binding domain and phosphorylation sites are absent in theTAL1-s protein translation product of this mRNA, while DNA-binding domains andthe helix-loop-helix domain are maintained. Furthermore, the third exon of theTAL1 comprises a highly conserved uORF sequence, an upstreamopen reading frame which acts as a cis-regulatory element inthe formation of TAL1 isoforms. The presence of uORF enables the initiation oftranslation, involving the eIF2 and eIF4E factors from the alternative siteslocated in exons 4–5 [14],producing a truncated form of the TAL1 protein.

Fig. 1

a. The structure of TAL1 gene – I–VI exons b. Long TAL1 transcriptvariant. Long isoform TAL1-L and short isoform TAL1-S are translated from thismRNA. UTR – untranslated mRNA region. uORF – upstream open readingframe. bHLH – mRNA region encoding the helixloop- helix domain. c. ShortTAL1 transcript variant from which only the TAL1-S isoform is translated

The truncated form TAL1-s is required for erythroid progenitorsdifferentiation, while the full-length protein TAL1-l is required formegakaryocytic differentiation of progenitor cells. It has been shown thattreatment of the human erythroid leukemia cell lines TF1 and HEL with erythroiddifferentiation inducers (DMSO and erythropoietin) produces not only theprimary (full-length) form of the TAL1-l protein, but also a truncated TAL1-sform [15]. It has been established thatsome anticancer agents acting on the components of the signaling pathwaysinvolved in the regulation of translation initiation may affect the TAL1-l toTAL1-s ratio. In particular, rapamycin (Rap,mTOR inhibitor) blocks theformation of truncated forms, while 2-Aminopurine (2AP, eIF2α-kinaseinhibitor) blocks the formation of full-length forms[14].

TAL1 FUNCTIONS IN EMBRYOGENESIS

The TAL1 transcription factor is essential for normal embryogenesis. Itsexpression starts on the 7^th day after fertilization, a day beforethe beginning of the development of circulatory system components. TAL1expression has been found in the blood islet cells of the yolk sac,endothelial cells, and angioblasts, and then in the liver and spleen of afetus, the major hematopoietic organs in embryogenesis. It has been shown thatthe cells involved in the formation of skeletal and nervous tissues alsoexpress TAL1 [16]. Inthe yolk sac and fetal liver, the Runx1 gene promoter andRunx3 gene enhancer are the major targets of TAL1[17]. Ets, GATA, and the Runx factor bindingsites, as well as a E-box sequence, have been found in the regulatory regionsof these genes. TAL1 and its partners GATA1, GATA2, E47, Ldb1, andLMO2 may form complexes at these DNA sites[18].Hematopoietic progenitor cells can also be derived fromhemogenic endothelial cells, a process that involves the Runx1 transcriptionfactor. TAL1 is required in order to produce hemogenic endothelial cells frommesoderm [19]. At later stages ofembryonic development, TAL1 regulates the differentiation of hematopoieticprogenitors into red blood cells, megakaryocytes, and platelets[20]. During embryogenesis, the cells that formblood vessels also express TAL1 [16].A lack of TAL1 expression not onlyresults in impaired hematopoiesis, but also in early embryonic death[2, 21].It has been demonstrated in a murine model that embryonic stem cells (ESCs) notexpressing TAL1 are not differentiated into hematopoieticcells under the action of hematopoietic differentiation factors[21]. Ectopic expression of TAL1in ESCs induces the formation of hematopoietic cells. In vitroexperiments have demonstrated that ESCs without TAL1expression are characterized by a low effectiveness of differentiationinto erythroid progenitor cells and cannot form colonies of lymphoid andmyeloid progenitor cells [22].

Fig. 2

The process of hematopoietic cell development during embryogenesis. Sometranscription factors defining the differentiation direction are shown abovethe arrows. ESC – embryonic stem cell, HSCP – hematopoietic stemcell progenitor

Thus, TAL1 directs the differentiation of hematopoietic progenitors at allthree stages of hematopoiesis during embryonic development. TAL1 acts on bloodprogenitor cells in the yolk sac (the first stage of hematopoiesis), determinesthe development and differentiation of hemangioblasts from their aggregation inthe primary strip until their migration into the hematopoietic islets of theyolk sac (the second stage of hematopoiesis). At the beginning of the thirdstage of hematopoiesis, TAL1 is required for hemangioblast differentiation inHSCs. It activates the expression of genes important for the maturation of erythroid,megacaryotic, and mast cells, and it is likewise involved in vascular systemremodeling (Fig. 2)[23].

THE ROLE OF TAL1 IN THE REGULATION OF HEMATOPOIESIS

In adults, mature blood cells are derived from pluripotent HSCs. HSCs areretained in the bone marrow during replication quiescence stage G0, due to theinteraction between their superficial cellular receptor protein (C-KIT, MPL,CXCR4) and the ligands on stromal cell surfaces[24, 25].The pluripotent HSCs respond to hematopoietic stress by terminating the quiescentphase and initiating active proliferation, receiving signals for furtherdifferentiation, and leading to the appearance of myeloid and lymphoidprogenitor cells. Some transcription factors essential to the hematopoiesisprocess are also the key factors in maintaining HSCs in the quiescent stage.These include the TAL1, E47, GATA2, and Ldb1, LMO2 components of theSCL complex [26]. Transformation ofKLS+/CD150+/CD48+ HSCs from the quiescent phase G0 to stage G1 is assisted bythe cyclin-dependent kinase P21/CDKN1A. TAL1 blocks this transition, increasingthe expression of the P21/ CDKN1A inhibitor [27]. Simultaneously, TAL1 enhances the expression of thetranscription factor ID1. Importantly, TAL1 does not belong to the proteinsessential for the survival and self-renewal of HSCs [3]. Its related protein LYL1 supports HSC survival in the caseof TAL1 knockout [28].Interestingly, TAL1 plays the opposite role in cord blood HSCs, where it,contrarily, activates G0–G1 transition, which is regulated using the mTORsignaling pathway [29]. However, TAL1and LYL1 are not interchangeable in differentiation processes and both proteinsare required for normal erythropoiesis and the formation of B-cells,respectively [30]. Unlike HSCs, TAL1functions as a cell cycle activator in myeloid and lymphoid progenitors,inhibiting the expression of the cyclin-dependent kinase inhibitors p21 and p16/Ink4a[31, 32].The hematopoietic transcription factors TAL1, GATA2, and LMO2, whose expressionlevel differs in each cell type, regulate the process of blood cell differentiation andmaturation (Fig. 3)[33]. The expression ofTAL1 is not identical in all hematopoietic cells. Highexpression levels of this gene have been detected in HSCs, in myeloidprogenitors, and in some mature myeloid cells (megakaryocytes, erythrocytes,mast cells, and basophils). Low levels of TAL1 are characteristic of lymphoidprogenitors, eosinophils, macrophages, and neutrophils[34-36].Mature T- and B-cells do not express TAL1 [37].Certain genes specific to erythroid cells are activated by a complex formed by GATA1 and TAL1[38].

Fig. 3

Adult hematopoiesis scheme with some key transcriptional factors. HSC –hematopoietic stem cell, MPP – multipotential progenitor, CMP –common myeloid progenitor, CLP – common lymphoid progenitor, MP –megakaryocyte progenitor, EP – erythropoietin progenitor, EBP –eosinophil–basophil progenitor

An analysis of the ChIP-seq has shown that TAL1 controls both the processescommon to all cells (cell cycle regulation, proliferation, apoptosis) and thosespecific only to erythroid cells (redox processes, heme biosynthesis,organization of the cytoskeleton), which is indirectly indicative of itsmultifunctionality [39]. In myeloid andlymphoid progenitor cells, the genes that control proliferation and apoptosisplay the role of TAL1 targets. Additionally, the pattern of TAL1 binding totarget genes widely varies with cell maturation. The dynamic changes inTAL1 expression suggest that the TAL1 factor demonstratesdiffering activity in cells during the initial choice of differentiationdirection and formation of mature blood cells, while its multifunctionality isdirectly related to its ability to form multicomponent complexes in theregulatory regions of target genes [8].There is evidence that the role of TAL1 in the differentiation of erythroidcells is effected, among others, using caspase-3, inducing cleavage of thisprotein. It has been shown that its activity eventually leads to a decrease inthe expression of GATA1 and BCL-XL, therebyinducing apoptosis in these cells [40].Some amino acid residues of TAL1 may undergo phosphorylation. For example, inerythrocytes, Akt kinase phosphorylates Thr 90 in TAL1. This modificationreduces the ability of TAL1 to repress the EPB42 genepromoter, whose product, the 4.2 protein, is required to build the erythrocytecytoskeleton [41]. The Ser172 residuemay also be phosphorylated by the cAMP-dependent protein kinase (PKA), whichaffects TAL1 binding to the E-box in the regulatory sites of various genes[42].

SCL-COMPLEX: ITS COMPONENTS AND TARGETS IN NORMAL HEMOPOIESIS

The proteins involved in normal hematopoiesis (LMO2, Ldb1–2,Gata1–3, Lyl–1, E2A/HEB, Runx1, ETO2, ERG, FL1) are themain partners of TAL1 in hematopoietic cells(Fig. 4).TAL1 directly binds to the LIM-domain of the LMO2 protein, which, inturn, interacts with Ldb1. LMO2 has no DNA-binding domain and actsas a bridge factor, which complexes TAL1 with other transcription factors inhematopoietic cells[43, 44].It may also form an extended complex, binding ETO2, RUNX1, ERG, or FLI1[45]. E-proteins (E12, E47), containinghelix-loop-helix domains, are required for TAL1 binding to the E-box sequences(CANNTG) in the regulatory regions of genomic DNA. In the complex, TAL1 regulatesthe activity of certain signaling pathways during the differentiation of hematopoieticcells. For example, TAL1 is essential for the survival of hematopoieticprecursors cultured in the presence of SCF, a ligand of the receptor tyrosinekinase C-KIT, which plays an important role in hematopoiesis[46]. The main role of the SCL complex inC-KIT regulation is associated with its ability to bind the promoter of this gene.It has also been established thatcomponents of the SCL complex may bind to variouscomponents of the C-KIT signaling pathway and change its activity[46-51].

Fig. 4

TAL1 and its partners involved in the differentiation, proliferation, andsurvival of hematopoietic cells

Furthermore, there is a direct correlation between the level of TAL1expression and phosphorylated forms of MEK and ERK1/2 kinases, thecomponents of the MEK/ERK signaling pathway[40]. In hematopoietic cells, the activityof MEK and ERK1/2 kinases is associated with the differentiation of myeloid,erythroid, and megakaryocytic hematopoietic cells[52].TAL1 probably participates in the differentiation of CD34+ hematopoietic cellsthrough the MEK/ERK signals[52, 53].

THE FUNCTIONS OF THE SCL COMPLEX AND ITS INDIVIDUAL COMPONENTS IN CARCINOGENESIS

As noted above, the normal level of TAL1 expression inlymphoid cells is much lower than that in myeloid ones [37]. Enhanced expression of TAL1 in T-cellsoften leads to their malignant transformation. Abnormally high expression ofTAL1 can result from chromosomal rearrangements, deletions,and mutations affecting the gene [54].The chromosomal translocation t (1; 14) (p32; q11) was found in 3% of cases ofT-cell leukemia. The chromosomal translocation t (1; 14) (p32; q11), leading tothe formation of the TRA/TAL1 fusion gene, was detected in 3% of cases ofT-cell leukemia. Deletion of 90 bps between the 5`-noncoding region of theTAL1 gene and SIL gene results in theformation of a SILTAL1 fusion gene controlled by theSIL gene promoter [54].The expression level of SIL in T-cells is normally very high,and, therefore, this translocation results in a high expression ofthe SIL-TAL1 fusion gene[55]. This deletion has been detected in20–25% of patients with T-ALL [54,56, 57].However, in most TAL1-positive cases of T-cell leukemia, an abnormally high expressionof TAL1 is effected without the participation of chromosomalrearrangements. Along with a high expression ofTAL1, significant expression levels of TLX1and LMO2were detected in most primary T-ALLsamples [58]. Increased activity ofTAL1 in T-cells results in an increased lifetime for lymphoidcells in the form of immature thymocytes. It is assumed that this can beconsidered as an event initiating the development of T-cell leukemia[59].

In T-ALL cells, TAL1 preferably binds to CAGGTG E-box sequences. AlthoughGATA1–3 factors often serve as intermediaries in TAL1 binding to theregulatory sites of DNA in T-cell leukemia cells, there are alternative bindingsites, in particular Runx and Ets [59].It has been shown that the TAL1 transcription factor directly activates theexpression of Runx1, Ets1, and GATA3in the blast cells of patients with T-ALL[60]. Furthermore, the GATA3 and Runx1factors enhance the expression of the TAL1 gene, which may indicatethe need for a positive feedback loop for the abnormal expression of the factorsinvolved in blood cell malignant transformation. In 45% of cases of TAL1-positiveleukemia, LMO1 and LMO2 mutant proteins formed due to chromosomalrearrangements of their encoding genes were detected[61]. Expression of all these factors leadsto the fact that double negative (CD4-CD8-) preleukemic thymocytes become capable ofdivision. Additionally, the Notch signaling pathway, whose components are involved inthe accumulation of mutations and impairment of differentiation processes, is oftenactivated in these cells. This leads to initiation and progression of T-cellleukemia [62]. In the case of malignanttransformation , TAL1 is often involved in the abnormal transcription ofvarious genes. In this case, as in normal hematopoiesis, it forms complexeswith the hematopoietic factors LMO2, Ldb1, and E12/E47[46,47].It has been established that overexpression of TAL1 and LMO2 isoften observed in T-ALL cells. Normally, LMO2 and TAL1 independentlyregulate the transcription of their own target genes, but they cooperativelydisrupt the functioning of the E2A factor in T-ALL cells, which contributes tothe development of leukemia[63, 64].It has been shown that the transcriptionfactor FOXP3 can act as a tumor suppressor in T-cell leukemia. It binds toLMO2 and reduces the likelihood of it interacting with TAL1,resulting in reduced transcriptional activity of the TAL1/LMO2complex [65].

Fig. 5

The structure of the SCL complex in the promoter region of the receptortyrosine kinase C-KIT gene

C-KIT receptor tyrosine kinase is one of the main targets of TAL1[48, 66].Hematopoietic progenitor cells are characterized by ahigh expression level of TAL1 and C-KIT. Ithas been shown that ectopic expression of TAL1 results in theinduction of C-KIT expression in B-lymphocytes, which normallydo not express these genes [66]. Somehematological malignancies, including acute myeloid leukemia and chronicmyeloid leukemia, are associated with an abnormally high expression ofC-KIT. The SCL complex acts as a specific activator receptor tyrosinekinase C-KIT gene promoter(Fig. 5).All the components of the complex (TAL1, LMO2, Ldb1, GATA2, E47) arerequired for it to function at its maximum. Studies in a murine embryonicfibroblast model have shown that the transcription factors E47 and GATA, takenalone, do not affect the activity of the C-KIT gene promoterdespite the fact that they activate the transcription of many genes in humanhematopoietic cells [66]. The samemurine system was used to show that the promoter is only activated in the caseof formation of a multicomponent complex whose main component is TAL1. GATA1and GATA2 are interchangeable: however, the complex comprising GATA1 possess alower transcriptional activity. The Sp1 protein, comprising zinc fingers andbinding GC-rich sequences, is also required to form the active SCL-proteincomplex. It has been shown that removal of E-box and GATA from the promoterregion of C-KIT does not reduce the activating activity of theSCL complex. Probably, Sp1 is also involved in attracting complex components tocertain target genes.

CLINICAL SIGNIFICANCE OF TAL1

Fig. 6

Possible partners of TAL1 and their interactions in normal and malignanthematopoietic cells

The extensive body of evidence of TAL1 participation in the development ofT-cell leukemia suggests that inhibitors of this protein, as well as inhibitorsof the associated signaling cascades, can be used as promising therapeuticagents to treat leukemia characterized by an abnormal activity of TAL1. At themoment, novel low-molecular-weight inhibitors of TAL1 are being developed andsynthesized in many laboratories. However, sufficiently strong and specificinhibitors of this protein have not been achieved so far. Phosphorylation ofTAL1 with MEK/ERK kinases is required to effect its transcriptional activity.The prospects of using the inhibitors of MAPK/MEK/ERK signaling pathwaycomponents as potential therapeutic targets are being discussed[67]. At the same time, there is evidence thattreatment of a mesenchymal stromal cell culture (stromal components of the bonemarrow) with MEK inhibitors results in the secretion of proinflammatorycytokine interleukin-18 by these cells[68]. This improves the survival chances ofT-ALL blast cells. The potential TAL1 protein targets associated with the implementationof its transcriptional activity are considered as promising targets for the therapy ofTAL1-associated T-cellleukemia (Fig. 6).These proteins include UTX demethylase (also known as KDM6A). It has been shown thattreatment of TAL1-positive blast cells with the T-ALL UTX inhibitor reduces the rateof their proliferation and stimulates apoptosis[69]. It has been determined that the use ofHDAC histone deacetylase inhibitors leads to a decreasein TAL1 expression and induces the apoptosis of blast cells of T-cellleukemia [70]. At the moment, the stoichiometryof the SCL complex is being actively explored. The results of such studies areexpected to open up new possibilities for the development of highly effectivetherapeutic agents targeting TAL1-positive leukemia, which could act byinterfering with the protein-protein interactions between the components of theSCL complex but not affect the viability of normal hematopoietic cells[41].