Cell and Molecular Biology of Human Leukaemias

 

Essentials

The human leukaemias arise when haematopoietic stem and progenitor cells acquire genetic alterations that lead to malignant transformation, following which affected cells can exhibit differentiation arrest in any lineage and at any stage of maturation.

Genetic alterations leading to leukaemia—a recurring theme is that the genes most frequently altered are those with evolutionarily conserved roles in the embryological development of various cell lineages and organ systems, including (but not limited to) genes that control normal haematopoiesis. The molecular genetic alterations that drive leukaemogenesis can generally be characterized into lesions affecting transcription factors and those that aberrantly activate signal transduction pathways, which often occur via activating mutations in tyrosine kinases.

The effects of leukaemogenic genetic alterations—some of these affect cell proliferation or survival, whereas others exert their primary effects on cell differentiation. The critical lesion almost always involves a ‘master’ transcriptional regulatory gene or tyrosine kinase signalling molecule that stands near the top of a hierarchy of gene control, such that leukaemia is efficiently instigated by a limited number of alterations rather than by multiple changes affecting tens of responder genes in the biochemical cascade.

Cell and molecular biology of particular leukaemias—(1) Acute lymphoblastic leukaemia and acute promyelocytic leukaemia—in most cases the initial lesion appears to affect progenitors at the same stage of differentiation as the predominant phenotype in the malignant clone. (2) Acute myeloid leukaemia and chronic myeloid leukaemia—most cases appear to arise in a primitive haemopoietic stem cell rather than a committed myeloid progenitor, with subsequent blockade of differentiation at a later developmental stage that determines the morphological subtypes of myeloid leukaemia apparent at diagnosis; this may also be the situation in some cases of acute lymphoblastic leukaemia, at least those expressing the BCR-ABL1 fusion oncogene. (3) Chronic lymphocytic leukaemia—it is currently believed that defective apoptosis in vivo is the primary pathogenic abnormality.

Introduction

Attempts to understand the pathobiology of the human leukaemias have focused on clinical presentation, cell morphology, histochemistry, cell immunophenotype, cytogenetics, and, in recent years, molecular genetics. Clinical and cell-biological findings are useful in diagnosis and risk assessment; however, most insights into the mechanisms underlying leukaemic transformation have relied on analysis of genes involved in recurrent cytogenetic and molecular genetic abnormalities. The emerging picture of leucocyte transformation indicates that most cases of leukaemia involve chromosomal translocations that result in aberrantly expressed transcription factors or activated tyrosine kinases, which drive malignant conversion and maintain the leukaemic phenotype. Transcriptional control genes are frequently activated by chromosomal translocations, which lead either to the production of structurally abnormal transcription factors generated by the fusion of disparate gene fragments, or to the aberrant expression of structurally intact genes that have been placed in the vicinity of transcriptional control elements (promoters and enhancers) that are normally highly active in the cell that subsequently becomes transformed: for example, the T-cell receptor (TCR) or immunoglobulin (IG) gene regulatory elements in lymphoid leukaemias. Oncogenic transcription factors appear to act by either up-regulating critical target genes that co-ordinate normal cell proliferation or survival (gain of function), or by interfering with normal regulatory cascades controlling programmed cell death and differentiation (loss of function). Similarly, signal transduction pathways, which are typically activated through point mutations in receptor tyrosine kinases or other genes mediating growth factor receptor signalling, act through signal transduction cascades that ultimately dysregulate the transcriptional control of gene expression. This article provides an introduction to the cell and molecular biology of leukaemia, focusing on characteristic examples of molecular genetic alterations that drive malignant transformation in the human leukaemias. Whenever possible, we have attempted to link advances in the molecular biology of a particular leukaemia to unique implications for treatment and prognosis.

Acute lymphoblastic leukaemia (ALL)

Approximately 5000 people in the United States, two-thirds of them children, develop acute lymphoblastic leukaemia (ALL) each year. More than 80% of children who are older than 12 months of age at ALL diagnosis achieve long-term event-free survival with modern risk-adapted therapy, attesting to the remarkable treatment advances that have been made over the past three decades. Unfortunately, fewer than 50% of adults become long-term survivors. The marked differences in outcome across age groups may be explained in part by differences in the age-specific incidence of genetic features that have prognostic implications. For example, the TEL-AML1 translocation, which is associated with an excellent prognosis, is detectable in 25% of children with ALL but is rare in infants and adults, while the BCR-ABL1 chimaeric oncogene, which is associated with poor responses to conventional chemotherapy, is much more common in adults. Despite differences in the incidence of several molecular genetic abnormalities across age groups, most of these abnormalities occur in both children and adults, and data is accumulating that similar molecular defects share similar pathophysiology across age groups.

In most instances, the pathobiology of transformed B-lymphoid cells mirrors the altered expression of genes that contribute to the normal functioning of developing lymphocytes, or occasionally mature B cells, although it may involve the aberrant expression of normally quiescent genes. Approximately 80% of patients with ALL have lymphoblasts whose phenotypes correspond to those of B-cell precursors. Only 2 to 3% of these patients have mature B-cell leukaemia, which is thought to represent a disseminated form of Burkitt lymphoma.

B-lineage ALL

BCR-ABL1

The first constitutively activated tyrosine kinase described in ALL results from fusion of 5′ sequences of the BCR proto-oncogene to 3′ sequences of the ABL (also known as ABL1) kinase due to action of the t(9;22) translocation, leading to creation of the so-called Philadelphia (Ph) chromosome. Most investigators believe that the t(9;22) translocation occurs in haemopoietic stem cells possessing both lymphoid and myeloid differentiative potential. Differences in the incidence of BCR-ABL1+ leukaemias between children and adults are striking. Found in only 5% of newly diagnosed paediatric ALL cases, this translocation is present in at least 25% of adult cases. ABL encodes a nuclear protein with tyrosine kinase activity, the normal function of which remains incompletely understood. However, it may stimulate p53-dependent growth arrest, suggesting a role as a cell cycle checkpoint after damage to the cellular genome.

The BCR-ABL1 translocation occurs both in ALL and in chronic myelogenous leukaemia (CML). In CML, the BCR breakpoints on chromosome 22 occur almost exclusively in the M-bcr (major breakpoint cluster region). In ALL, the BCR breakpoints tend to cluster in the m-bcr (minor breakpoint cluster region), although they also occur in the M-bcr. Breaks in the m-bcr region lead to fusion genes encoding a 190-kDa protein (p190), while those in M-bcr generate a 210-kDa protein (p210). Both the p190 and p210 forms of BCR-ABL1 are localized in the cytoplasm, have increased tyrosine kinase activity, and can transform haematopoietic cells in experimental systems. The precise mechanism by which BCR-ABL1 transforms human haemopoietic cells is incompletely understood, but this fusion kinase has been shown to activate several SRC family kinases and activate the RAS, PI3K, and JAK/STAT signal transduction pathways.

The expression of a BCR-ABL1 fusion gene is one of the few situations in which bone marrow transplantation for patients with ALL in first remission is clearly beneficial. Additionally, several kinase inhibitors have been identified that target the ABL tyrosine kinase and are highly active in BCR-ABL1+ CML. Although imatinib, the first of these targeted kinase inhibitors, shows activity against BCR-ABL1+ ALL, success rates with this as a single agent in ALL have been disappointing, as resistance frequently emerges via mechanisms discussed in the CML section below. However, the addition of imatinib to standard chemotherapy has led to remarkable improvements in short-term event-free survival in pediatric BCR-ABL1+ ALL, raising the possibility that this combination may supplant the need for bone marrow transplantation in first remission.

Tel-aml1

The TEL-AML1 (also termed ETV6-RUNX1 or ETV6-CBFA2) translocation is present in 25% of cases of childhood ALL; however, it is not detectable by standard cytogenetic analyses and thus was discovered only relatively recently. The TEL-AML1 translocation often arises prenatally and is probably the initiating lesion in ALL, although it is insufficient to cause leukaemia, because the incidence of detectable TEL-AML1 translocations in normal newborns is approximately 100-fold greater than the incidence of ALL. This chimaeric oncogene is generated by a t(12;21) translocation, which leads to the fusion of the HLH domain of TEL to almost all of AML1. Both of these genes are transcription factors required for normal haematopoiesis, and both are involved in other leukaemia-related translocations. However, the precise oncogenic mechanisms mediating transformation by TEL-AML1 remain incompletely understood.

The identification of the TEL-AML1 translocation, which occurs almost exclusively in children, is associated with a particularly favourable prognosis, with event-free survival rates of approximately 90%. These cases appear to be particularly responsive to antimetabolite chemotherapy, perhaps as a result of decreased expression of the MDR-1 multidrug resistance gene and of genes involved in purine biosynthesis. The presence of the TEL-AML1 fusion identifies a large group of children that are excellent candidates for less intensive therapy.

E2A fusion genes

The E2A gene, which encodes a bHLH transcription factor on chromosome 19, is targeted by two recurrent translocations in patients with ALL—the t(1;19), found in 3 to 5% of all cases, and the t(17;19) found in approximately 0.5% of children. The former generates one of the best-characterized fusion oncogenes in ALL, in which the transcriptional transactivation domains of E2A are linked to the DNA-binding homeodomain of PBX1. PBX1 is an orphan HOX gene that shares homology with the exd gene of Drosophila, which regulates segment identity through direct interaction of its product with specific HOM proteins of the Bithorax and Antennapedia complexes.

E2A-PBX1, which binds to DNA in a site-specific manner, is clearly oncogenic in fibroblast transformation assays, and appears to induce programmed cell death (apoptosis) in lymphoid cells. Like its exd homologue, PBX1 is directed to its consensus DNA-binding site by a subset of interacting HOX proteins, whether or not it is fused to E2A. Surprisingly, these site-specific recognition sequences of PBX1 are not required for the transforming activity of E2A-PBX1. How, then, would the chimaeric oncoprotein induce leukaemia, if not by disruption of gene expression normally regulated by HOX proteins? The answer appears to lie in a short peptide sequence that regulates the HOX-specific protein–protein interaction, and which is essential for leukaemogenic activity. The presence of the E2A-PBX1 translocation was originally associated with a poor prognosis; however, this translocation is no longer associated with inferior outcomes in the setting of modern risk-adapted therapy for childhood ALL.

The t(17;19) translocation generates the E2A-HLF fusion gene, consisting of the N-terminal transactivation regions of E2A and the C-terminal DNA-binding and dimerization domains of HLF, a member of the bZIP transcription factor gene family. E2A-HLF appears to mediate leukaemic transformation largely through the transcriptional activation of SLUG, which inhibits p53-mediated apoptosis. The E2A-HLF translocation, which occurs in less than 1% of cases of ALL, is typically seen in adolescents, associated with hypercalcaemia and disseminated intravascular coagulation at the time of diagnosis, and appears to have an unfavourable prognosis, perhaps as a result of SLUG-induced resistance to chemotherapy.

MLL fusion genes

Translocations involving the MLL (mixed lineage leukaemia) gene, located on chromosome band 11q23, are found in 80% of cases of infant leukaemia, whether ALL or acute myeloid leukaemia (AML), and in 5 to 10% of primary ALL and AML cases in older children and adults. Furthermore, MLL translocations occur in 85% of secondary AML cases that develop in patients treated with topoisomerase II inhibitors. The MLL protein is expressed in all haematopoietic stem and progenitor cells and is required for normal haematopoiesis. It contains multiple domains, including three N-terminal AT hook DNA-binding motifs, a region with homology to DNA-methyltransferase, a transcriptional activation domain, and a C-terminal SET domain. The stability and localization of the MLL protein depend on proteolytic processing by Taspase1, a specialized protease that cleaves the MLL protein into N- and C-terminal fragments that remain associated through intramolecular protein–protein interactions. MLL binds DNA in a non-sequence-specific manner as part of large multiprotein complexes, and appears to act largely as a transcriptional activator.

More than 30 discrete chromosomal sites participate in 11q23 translocations, most commonly 4q21, 9p22, and 19p13, resulting in fusion of the AF4, AF9, and ENL genes to MLL. The leukaemogenic role of MLL fusions has been definitively demonstrated through the generation of mouse models, with mice expressing MLL-AF9, MLL-ENL, MLL-ELL, or MLL-CBP in their haematopoietic precursor cells developing leukaemias. As with PBX1, the dysregulation of HOX genes appears to play a prominent role in the pathogenesis of MLL-rearranged leukaemia. MLL is known to bind to and activate several HOX gene promoters, and leukaemias with MLL rearrangements characteristically show overexpression of specific HOX genes, including HOXA9, HOXA10, HOXC6, and the HOX gene regulator MEIS1. In particular, HOXA9 has been shown to play critical functions in the transformation of haematopoietic precursors by MLL fusion oncogenes in murine models of leukaemia.

The MLL-AF4, MLL-AF9, and other MLL fusion genes predict a dismal outcome in patients with early B-lineage ALL treated with conventional therapy. Additionally, allogeneic bone marrow transplantation to intensify therapy for infants with MLL-rearranged ALL in first remission is not clearly beneficial, and limited evidence suggests that this may actually worsen outcomes when compared with chemotherapy alone. One notable exception to the worse risk implications of MLL translocations is the t(11;19)(q23;p13.3) translocation leading to the MLL-ENL fusion in the case of T-ALL. Although this translocation is associated with poor prognosis in B-precursor ALL, the largest case series published to date shows a surprisingly good outcome in T-ALL patients treated with chemotherapy alone whose leukaemic cells harbour this translocation.

Myc

Chromosomal translocations in B-lineage cells can also mobilize proto-oncogenes to sites adjacent to normally active enhancer or promoter elements of IG genes, without structurally altering the oncogene involved. The prototype for this mechanism in B-lineage ALL is the t(8;14), which arises in mature B cells and places the MYC proto-oncogene on chromosome 8 under the control of IG heavy-chain gene regulatory sequences on chromosome 14. Similar repositioning of MYC adjacent to the light-chain regulatory sequences results from the t(2;8) or the t(8;22), which is seen in a much smaller percentages of cases. Through one of these translocations, MYC expression becomes highly up-regulated, leading to abnormally increased amounts of the MYC protein, a transcription factor that forms a DNA-binding complex with another cellular protein (MAX), and eventually leads to disruption of gene expression involved in the control of cell proliferation.

Although the coding region of MYC is not structurally altered by these translocations, point mutations commonly arise in these cases at codons 58 or 62 of the MYC protein. These point mutations disrupt phosphorylation sites involved in regulating the activity and stability of the MYC protein and not only lead to the accumulation of activated and stabilized MYC protein, but also impair the ability of MYC to activate an important tumour suppressor mechanism mediated by the BIM gene.

Children and adults with MYC-translocated mature B-cell ALL have extremely poor outcomes when treated with conventional regimens designed for the treatment of ALL. However, these patients have excellent responses to the relatively brief but intensive regimens designed for the treatment of Burkitt lymphoma, and most experts now consider mature B-cell ALL to be a disseminated form of Burkitt lymphoma. Thus, B-cell ALL was the first example of a subtype of ALL requiring tailored therapy with a vastly different drug regimen for effective disease control.

T-lineage ALL

First recognized as a distinct clinical entity in the early 1970s, T-ALL accounts for 10 to 15% of acute lymphoblastic leukaemias in children and 20 to 25% of cases in adults. The disease can arise in thymocytes at any stage of maturation, defined on the basis of reactivity with monoclonal antibodies (CD4/CD8 double-negative immature thymocytes, cytoplasmic CD3+, CD7+, CD2+, and CD5+; CD4/CD8 double-positive common thymocytes, cytoplasmic CD3+, CD1+, CD2+, CD5+, CD7+, CD10+, CD4+, and CD8+; and CD4/CD8 single-positive late thymocytes, cytoplasmic CD3+, CD2+, CD5+, CD7+, CD4+, or CD8+).

Dysregulated expression of oncogenic transcription factors

In contrast to the fusion oncogenes that drive the development of B-cell precursor ALL, oncogene activation in T-ALL typically reflects the overexpression of genes encoding structurally intact T-cell proto-oncogenes. Approximately 25% of cases of T-ALL have cytogenetically identifiable chromosomal translocations that typically mobilize intact proto-oncogenic transcription factors into the vicinity of transcriptionally active sites of the β or α/δ loci of the T-cell receptor genes (TCRβ or TCRα/δ). Among the genes that are aberrantly expressed in thymocytes and cause leukaemic transformation through this mechanism are those representing the bHLH family of transcription factors (TAL1/SCL1, TAL2/SCL2, LYL1, and BHLHB1), the bHLH/ZIP family (MYC), other nuclear regulatory proteins (LMO1 and LMO2), homeobox proteins (HOX11), and a truncated and constitutively activated form of NOTCH1. The relationship of these genes to the pathogenesis of T-ALL has been established by their recurrent involvement in translocations that affect thymocytes or their precursors. Surprisingly, many of the T-cell oncogenes identified to date are not usually expressed in T cells; hence, their ability to induce leukaemia most likely reflects the misexpression of master transcriptional control genes with the disruption of normal T-cell developmental pathways. This is illustrated by HOX11, which is not normally expressed in lymphoid cells, but has been shown to be essential for normal development of the spleen.

Despite intensive cytogenetic research, chromosomal translocations have been identified in only about 25% of T-ALL cases. However, gene expression profiling has identified overexpression of many of the oncogenes found in recurrent T-ALL chromosomal translocations in most cases of ALL, even when such translocations are not present, suggesting that translocation-independent mechanisms are at work that disrupt these key transcriptional control networks in thymocyte development, leading to overt T-ALL.

NOTCH1 mutations

The NOTCH1 gene was initially identified as a partner gene in a t(7;9) translocation in very rare cases of T-ALL, in which a truncated NOTCH1 is placed under control of the T-cell receptor β locus. Despite the rarity of this translocation, a search for activating mutations in NOTCH1 identified these in greater than 50% of cases of T-ALL. NOTCH1 encodes a cell surface receptor that, upon ligand binding, undergoes proteolytic processing that culminates in the proteolytic release of an intracellular portion of the receptor known as intracellular NOTCH1 (ICN), which then moves to the nucleus where it is active as a transcription factor. The NOTCH1 mutations found in T-ALL either lead to the ligand-independent proteolytic release of the ICN, or mutate the PEST domain of the ICN, thus leading to accumulation of nuclear ICN protein as a result of impaired proteolytic degradation.

The identification of NOTCH1 mutations in T-ALL has generated considerable excitement for the application of γ-secretase inhibitors to the treatment of patients with T-ALL. γ-Secretase is required for the proteolytic activation of NOTCH receptors, and inhibitors of this enzyme, which have previously been developed due to the role of γ-secretase in Alzheimer’s disease, effectively inhibit NOTCH receptor activation in experimental systems. Such γ-secretase inhibitors (GSIs) are currently undergoing clinical trials in patients with T-ALL. Although GSIs are effective against several human T-ALL cell lines harbouring activating NOTCH1 mutations, some of these cell lines are resistant. Recent data demonstrate that the effect of GSIs in cultured T-ALL cell lines requires the PTEN tumour suppressor, and PTEN loss mediates resistance to these inhibitors in cultured T-ALL cells. Interestingly, pharmacological suppression of the AKT pathway, which is normally inhibited by PTEN, was effective against PTEN-null, NOTCH1-mutated T-ALL. Moreover, recent data has demonstrated that GSI therapy reverses glucocorticoid resistance in T-ALL cells, while glucocorticoids simultaneously prevent GSI-induced gastrointestinal toxicity in the mouse. Taken together, these data suggest that therapy with a NOTCH1 inhibitor, an AKT pathway inhibitor, and a glucocorticoid may prove to be a particularly effective combination in T-ALL. Ongoing studies to clarify the molecular pathogenesis of T-ALL should greatly enhance the value of molecular genetics in predicting the clinical responses of patients with T-ALL and, ultimately, could provide additional effective targets for novel drug therapies.

Lesions activating tyrosine kinases, RAS, and PI3K signalling pathways in ALL

Oncogenic lesions leading to the aberrant activation of signal transduction pathways that normally mediate growth factor signalling are common events in both B-precursor and T-ALL. As previously described, the BCR-ABL1 fusion oncogene, found in t(9;22)-translocated B-precursor ALL and chronic myeloid leukaemia, acts as a constitutively activated tyrosine kinase to activate a number of growth factor signalling pathways. Although BCR-ABL1 fusions are rare in T-ALL, NUP214-ABL1 fusions have recently been described in 6% of patients with T-ALL. These fusions arise via a mechanism in which a portion of chromosome band 9q34, which contains both NUP214 and ABL1, is circularized in a manner leading to the fusion of these 2 genes, with the same breakpoint in ABL1 as is seen in the BCR-ABL1 translocation. Similar to the BCR-ABL1 fusion oncoprotein, the NUP214-ABL1 fusion has constitutive ABL1 tyrosine kinase activity that can be inhibited by the BCR-ABL1 kinase inhibitor imatinib.

FLT3 encodes a receptor tyrosine kinase that plays important roles in early haematopoietic precursors. Although activating mutations in this gene are rare in adult ALL, FLT3 is commonly mutated or overexpressed in most cases of ALL that involve MLL gene rearrangements or hyperdiploidy, which are typically seen in childhood ALL. FLT3 mutations also occur in AML and are discussed in more detail in that section below.

The RAS guanine nucleotide-binding proteins, which were originally identified due to their homology with viral oncogenes, are activated by haematopoietic cytokine receptors in response to ligand binding in normal haematopoietic precursors. Activating mutations of NRAS are found in approximately 10% of cases of ALL, while KRAS mutations occur in 5 to 10%. These mutations typically consist of single amino acid substitutions at codons 12, 13, or 61 of RAS, and lead to constitutive receptor-independent activation of downstream signal transduction pathways, including the MAPK and PI3K pathways. Activation of the PI3K pathway in T-ALL can also occur via loss of the PTEN tumour suppressor, an abnormality which is found in approximately 15% of cases of T-ALL.

Acute myeloid leukaemia (AML)

The estimated number of new cases of acute myeloid leukaemia (AML) occurring annually in the United States is vastly higher in adults than in children (20 000 vs 1000). Overall, prognosis remains poor in both age groups, although several cytogenetic and molecular genetic abnormalities have been found to have prognostic relevance and allow risk stratification into favourable, intermediate, and unfavourable subgroups. Additionally, evidence now suggests that bone marrow transplantation can improve outcomes for patients with higher-risk disease who have histocompatible donors and are young and healthy enough to tolerate this procedure. AML has traditionally been classified according to the developmental stage at which differentiation arrest occurs in the bulk of the myeloblast population, and differentiation stages in AML are best described in the context of the French-American-British (FAB) classification system. However, evidence now suggests that the transformed cell in AML is a haematopoietic stem or primitive progenitor cell that retains the ability to differentiate into the more mature myeloid cells that form the bulk of the tumour. Many cases of AML can now be classified based on the presence of characteristic chromosomal translocations, several of which have prognostic and therapeutic implications.

Recurrent genetic abnormalities in AML frequently target several transcription factors involved in the regulation of haematopoietic stem and progenitor cell differentiation, including the core-binding factor (CBF) transcription complex, RARα, MLL, and transcriptional coactivators such as CBP and MOZ. These genetic aberrations typically lead to the activation of self-renewal pathways, including the WNT-CTNNB1, NOTCH, and BMI1 pathways, and these are believed to function at the level of the leukaemic stem cells that maintain the myeloid leukaemic cell population.

PML-RARα fusion

Promyelocytic leukaemia, defined as the clonal expansion of transformed myeloid cells blocked at the promyelocyte stage of development, is characterized by the presence of the t(15;17) translocation that generates the PML-RARα fusion gene. RARα (retinoic acid receptor alpha) is a member of the nuclear hormone–receptor superfamily, functioning as a ligand-dependent, zinc-finger transcription factor with a critical role in normal myeloid cell differentiation. The PML transcription factor is a tumour suppressor that regulates several apoptotic pathways via interactions with p53 and other proteins. The fusion PML-RARα protein contains nearly all the key functional domains of each molecule, including the proteinprotein interaction motifs of PML and the DNA-binding, dimerization, ligand-binding, and transcriptional activation domains of RARα. The PML-RARα oncoprotein induces leukaemia by inhibiting, in a dominant fashion, the normal biological activities of both RARα and PML. The net effect is a blockade of differentiation with immortality and sustained proliferation among promyelocytes, the hallmark of acute promyelocytic leukaemia (M3 AML). Treatment of PML-RARα+ AML with pharmacological dosages of the RARα ligand all-trans-retinoic acid (ATRA) results in the release of corepressor complexes from the PML-RARα fusion protein, reversing the protein’s inhibitory activity and enabling the leukaemic promyelocytes to proceed to terminal differentiation. The unique specificity of ATRA for the underlying molecular lesion in M3 AML has allowed marked improvements in outcomes for patients with promyelocytic leukaemia.

AML1-ETO

The AML1-ETO fusion gene (also known as RUNX1-CBFA2T1) results from the t(8;21) translocation, which is found in 10% of AML cases overall, particularly those of the FAB M2 subtype. This fusion oncogene consists of the N-terminal portion of AML1, involving the entire RUNT homology domain that mediates binding to the promoters of several haematopoietic genes, and the C-terminal portion of ETO, which mediates interactions with the nuclear corepressor complex. Thus, the AML1-ETO fusion exerts its leukaemic activity by recruiting nuclear corepressor complexes to the promoters of genes that are normally activated by the AML1-CBFβ complex, whose function is essential for the development of all haemopoietic lineages.

Patients whose myeloid blasts carry the AML1-ETO translocation have been shown to have a relatively favourable prognosis, and respond more readily to chemotherapy regimens involving high-dose cytarabine than most other patients with AML. Since AML-ETO is a dominant-negative chimaeric transcription factor that relies on corepressor complexes, novel treatments that disrupt the formation, stability, or activity of such complexes, such as histone deacetylase inhibitors, might reverse the leukaemic phenotype, as seen with the use of ATRA in patients with acute promyelocytic leukaemia carrying the PML–RARα oncogene.

CBFβ–MYH11

The CBFβ-MYH11 fusion product, due to inv(16) or t(16;16), is seen in approximately 5% of newly diagnosed cases of AML. Once thought to be pathognomonic for cases with dysplastic eosinophilic precursors among myeloblasts and monoblasts (M4Eo subtype), this finding has since been made in acute myeloblastic leukaemia (M1 and M2 subtypes). These genetic rearrangements fuse most of CBFβ to a variable amount of the C-terminal α-helical rod domain of MYH11, a smooth-muscle, myosin heavy-chain protein that possesses both actin-binding and ATPase activity. Like AML1-ETO, the CBFβ-MYH11 fusion protein aberrantly associates with nuclear corepressor complexes and results in the recruitment of these corepressor complexes to the promoters of genes normally activated by the AML1-CBFβ complex. As with AML1-ETO, the expression of the CBFβ-MYH11 fusion confers an increased probability of achieving a sustained remission with high-dose cytarabine-containing chemotherapy regimens.

MLL fusion genes

Translocations involving the mixed lineage leukaemia (MLL) gene are found in both AML and ALL, and the pathobiology of MLL fusion genes was reviewed in the ALL section earlier in this chapter. Interestingly, recent work has shown that, in a mouse model of MLL-rearranged acute myeloid leukaemia, the introduction of the oncogenic MLL-AF9 fusion gene into committed granulocyte–macrophage progenitor cells leads these progenitors to re-activate a subset of genes that are highly expressed in normal haematopoietic stem cells, while their overall gene expression profile remains very similar to that of normal granulocyte–macrophage progenitors, suggesting that the primary transformed leukaemic stem cell in this disorder in humans may also be a committed progenitor rather than a pluripotent haematopoietic stem cell.

NPM mutations

Mutations in the nucleophosmin gene (NPM) were recently identified in approximately one-third of all primary cases of AML, and occur selectively in AML cases with a normal karyotype. The NPM gene encodes a protein that demonstrates nuclear-cytoplasmic shuttling and has a wide range of biologic activities. NPM mutations in AML occur in exon 12 and result in a frameshift involving the C terminus of the protein. Although several different frameshift mutations are found, these uniformly lead to disruption of at least one of the tryptophan residues at positions 288 and 290, which disrupts the protein’s nuclear localization signal. Additionally, these frameshift mutations also introduce an aberrant nuclear export signal, resulting in abnormal subcellular localization with very high cytoplasmic and low nuclear levels of nucleophosmin.

NPM acts as a chaperone for proteins, nucleic acids, and histones. It is required for the assembly of mature ribosomal complexes, and it is thought to regulate protein synthesis and cell growth through its effects on the ribosome pool. NPM also maintains genomic stability through the regulation of DNA repair and centrosome duplication during mitosis, and it interacts with the key cell cycle regulators CDK2-cyclin E. Finally, NPM interacts with p53 and ARF to regulate the ARF-MDM2-p53 pathway. Although alterations in any of these functions could represent the mechanism driving selection for NPM mutations during leukaemogenesis, the precise mechanism(s) mediating the oncogenic effect of these NPM mutations is a matter of intense ongoing investigation.

Mutations activating tyrosine kinase and RAS signalling pathways in AML

Activating mutations leading to constitutive activation of growth factor receptor signalling pathways are common events in AML, as in ALL. The RAS guanine nucleotide–binding proteins, which were originally identified due to their homology with viral oncogenes, are activated in response to haematopoietic cytokine receptors in response to ligand binding in normal haematopoietic precursors. Point mutations in codons 12, 13, or 61 of KRAS and NRAS are found in the leukaemic cells of 25% and 15%, respectively, of patients with AML. These point mutations are oncogenic due to their constitutive, growth factor–independent activation of downstream signal transduction pathways, including the PI3K, MAPK, and RALGDS pathways. The data on the prognostic relevance of RAS mutations in AML remain inconclusive.

Mutations in the KIT tyrosine kinase, which is the receptor for the stem cell factor (SCF) ligand, are found preferentially in AML cases harbouring t(8;21), inv(16), or t(16;16), occurring in approximately 25% of these cases. These mutations typically involve the extracellular domain of the receptor or the catalytic domain, and result in spontaneous ligand-independent receptor activation. Mutations in the extracellular domain lead to activation of the MAPK and PI3K signalling pathways, while catalytic domain mutations have been shown to constitutively activate PI3K and STAT3 signalling. KIT mutations are associated with increased relapse rates in patients with the otherwise prognostically favourable t(8;21), inv(16), or t(16;16). However, imatinib and several newer tyrosine kinase inhibitors, originally designed to target the BCR-ABL1 fusion kinase, have also been found to be effective inhibitors of KIT signalling, and these small molecules may prove to have a role in the therapy of KIT-mutant AML.

The FLT3 gene encodes a receptor tyrosine kinase that is highly expressed in haematopoietic precursors, where it plays important functional roles. Activating mutations in FLT3 are typically either internal tandem duplications (ITD) in the juxtamembrane domain, or point mutations or insertions in the second tyrosine kinase domain. These mutations lead to ligand-independent autophosphorylation and activation of downstream signal transduction pathways. ITD mutations are found in 25% of cases of AML, while mutations in the kinase domain are found in 10% of these patients. Several studies now demonstrate that FLT3 ITD mutations are associated with poor outcomes in AML, particularly in the setting of high FLT3 allelic ratio. Experimental evidence suggests that the pharmacological inhibition of FLT3 has important antileukaemic effects, and several small molecule inhibitors of FLT3 are currently under clinical development.

Chronic myeloid leukaemia

Most patients with chronic myeloid leukaemia (CML), both children and adults, harbour the classic t(9;22) translocation in myeloid cells, giving rise to the Philadelphia chromosome and the BCR-ABL1 fusion oncogene. The pathobiology of the BCR-ABL1 fusion oncoprotein, which also occurs in ALL, was discussed in that section of this chapter. In most untreated patients with CML, the resulting disease is biphasic, with an initial (chronic) phase that lasts 3 years on average, and a terminal (blast) phase that is highly refractory to therapy and is generally fatal within a median of 2 to 4 months. Allogeneic bone marrow transplantation is the only known curative treatment for CML. However, therapy for patients with CML who lack histocompatible donors or are otherwise not candidates for bone marrow transplantation was revolutionized with the introduction of imatinib, which has altered the natural history of this disease. Imatinib is a small molecule that inhibits BCR-ABL1 tyrosine kinase activity by targeting its ATP-binding site. Although experimental and clinical data suggest that BCR-ABL1 inhibitors are unlikely to be curative as single agents, long-term imatinib therapy can control chronic-phase CML for many years. Imatinib resistance can develop in chronic-phase CML, and it tends to develop particularly rapidly in late-stage CML and in BCR-ABL1+ ALL, typically through point mutations in the kinase domain that impair imatinib binding without disrupting kinase activity. Several newer ‘second-generation’ BCR-ABL1 inhibitors have now been developed, many of which also inhibit other kinases including the SRC family kinases, which are known mediators of BCR-ABL1 oncogenic signalling. Some of these newer inhibitors have been shown to target most imatinib-resistant BCR-ABL1 mutants, and indeed are active against CML after imatinib failure, although resistance to these second-generation inhibitors can also arise. Several novel small molecules targeting BCR-ABL1 via ATP-binding-site-independent mechanisms, or targeting a broader range of kinases, are currently undergoing clinical development in an effort to overcome drug-resistant BCR-ABL1 mutations.

Chronic lympocytic leukaemia (CLL)

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in Europe and North America, with an estimated incidence of 15 000 cases per year in the United States. Although rare before 50 years of age, the incidence of CLL rises relatively rapidly thereafter. The diagnosis of CLL rests on the identification of an absolute peripheral blood lymphocytosis (>5 × 109 cells/litre) and an appropriate immunophenotype, and patients can also develop lymphomatous involvement of lymph nodes or other tissues. Interestingly, 3.5% of healthy individuals older than 40 years have a small but identifiable clonal population of lymphocytes with an immunophenotype consistent with CLL, although the clinical significance of this finding remains unclear. Despite the frequency of CLL, relatively little is known about its molecular pathogenesis, in large part due to difficulties in culturing these cells in the laboratory and bringing them into mitosis for karyotyping, and the lack of a suitable animal model for indolent CLL. The accumulation of mature B cells that have escaped apoptosis and undergone cell cycle arrest in G0/G1 is the hallmark of CLL, and these cells overexpress several of the antiapoptotic BCL2 family members, including BCL2 itself, BCL-XL, and MCL1. It is currently believed that defective apoptosis in vivo is the primary pathogenic abnormality in CLL, and inhibitors of these antiapoptotic proteins, which effectively induce apoptosis in these cells, are currently under clinical development for the treatment of this disease.

Clinical summary

Several molecular genetic changes in leukaemic cells at diagnosis are sensitive markers of response to therapy, and therefore can be used as guides to treatment. Table 1 summarizes the clinical utility of recognized oncogenic transcription factors in the human leukaemias. Thus far, only a few specific lesions, the PML-RARα fusion gene in acute promyelocytic leukaemia, the BCR-ABL1 kinase in CML and ALL, the NUP214-ABL1 kinase in T-ALL, and the KIT and FLT3 mutations in AML have been productive targets for molecular-oriented therapy, but this number will likely increase as scientists continue to unravel the genetic mechanisms that transform normal blood cells and maintain leukaemic phenotypes.

 

Table 22.3.1.1 Clinical applications of common oncogenic transcription factors in the human leukaemias

 

Altered gene Leukaemia subtype* Risk of treatment failure Recommended treatment
ALL
TEL–AML1 (ETV6–EBFA2) Pro-B cell Low Well-tolerated chemotherapy (antimetabolites primarily)
E2A–PBX1 Pre-B cell Intermediate Intensive chemotherapy (genotoxic drugs and antimetabolites)
MYC B cell High Intensive chemotherapy (rotation of genotoxic drugs)
MLL–AF4 CD10– pro-B cell Very high Intensive chemotherapy or allogeneic stem-cell transplantation
BCR–ABL1 Pro-B cell (predominantly) Very high Allogeneic stem-cell transplantation
AML
AML1–ETO Acute myeloblastic leukaemia with maturation (M2 morphology) Intermediate Intensive chemotherapy (including high-dose cytarabine)
CBFβ–MYHII Acute myelomonocytic leukaemia with eosinophils (M4Eo morphology) Intermediate Intensive chemotherapy (including high-dose cytarabine)
PML–RARα Acute promyelocytic leukaemia (M3 morphology) Intermediate Intensive chemotherapy (including all-trans-retinoic acid and an anthracycline)

ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia.

* Subclassifications of AML are those of the French–American–British (FAB) cooperative group.

† As determined in standard programmes of chemotherapy (without haemopoietic stem-cell rescue). Treatment failure refers to either remission induction or remission maintenance, or both. The average rates of long-term, leukaemia-free survival in children and adolescents with ALL or AML range from 65–70 per cent and from 30–40 per cent, respectively.

‡ The choice of therapy is based on detection of the indicated fusion gene at diagnosis by cytogenetic analysis, Southern blotting, or RNA-polymerase chain reaction assays for chimeric mRNAs.