Pathobiology of Acute Lymphoblastic Leukemia

MYC in Mature B-Cell Acute Lymphoblastic Leukemia

The vast majority of cases of mature B-cell ALL (Burkitt leukemia) are characterized by a translocation that places one allele of MYC from chromosome 8 under the control of the regulatory elements of an Ig gene, either the heavy-chain gene on chromosome 14q32 or the κ or λ light-chain genes on chromosomes 2 and 22, respectively. These translocations are oncogenic because they result in lineage-specific overexpression of MYC, a prototypical basic helix–loop–helix oncogenic transcription factor. The mechanisms through which MYC exerts its potent oncogenic effects have been the subject of intense investigation. MYC has been estimated to regulate the expression of 15% of the genome and leads to the transcriptional activation of a large number of genes involved in cell division, growth, metabolism, adhesion, and motility. MYC also exerts post-transcriptional effects on gene expression by regulating microRNA expression and ribosome biogenesis. MYC exerts its transcriptional activity via the formation of heterodimers with its DNA-binding partner protein MAX. MYC–MAX heterodimers bind to canonical hexameric E-box DNA sequences (5′-CACGTG-3′) where they activate transcription. MAX can also heterodimerize with other bHLH proteins, including MAD, MXI-1 (MAD2), and MNT. Whereas transcriptional activation by MYC–MAX complexes promotes proliferation, binding by MAD–MAX and other MAX heterodimers produces opposite effects. For example, MAD inhibits MYC function both by competing with MYC for binding to MAX and by directly inhibiting transcription.

Recent work has revealed that a major consequence of MYC overexpression is the global amplification of gene expression. Investigation of transcriptional regulation mechanisms has revealed two distinct steps in the regulation of gene expression by transcription factors. First, RNA polymerase II and its associated transcriptional apparatus are loaded onto a gene promoter by one set of transcription factors, but RNA polymerase is often initially “paused” near the proximal promoter. Subsequent release from transcriptional pause is a distinct and highly regulated step in the control of gene expression. Unexpectedly, recent studies have revealed that a major consequence of MYC overexpression is the release of transcriptional pause at genes that were already loaded with RNA polymerase, rather than the recruitment of RNA polymerase to new target genes. These findings support a model in which MYC overexpression functions to amplify the expression of genes that are already being transcribed, thus “locking in” a cell’s existing transcriptional program. The acquisition of an MYC-activating lesion in a cell with a highly proliferative gene expression program that is normally transient, such as an immature B-cell progenitor, can lock this cell in this highly proliferative state, thus providing one mechanism to explain MYC-driven oncogenesis.

TAL1 and LMO Genes in T-Cell Acute Lymphoblastic Leukemia

In leukemia with a T-cell phenotype, the breakpoints of recurrent chromosomal translocations consistently juxtapose TCR gene-regulatory elements, which are highly active in committed T-cell progenitors, to the protein-coding sequence of oncogenic transcription factors, which then become aberrantly overexpressed as a result of these translocations. The best characterized of the oncogenic transcription factors involved is TAL1 (also known as SCL). TAL1 is overexpressed as a result of the recurrent t(1;14) translocation or intra-chromosomal deletions in approximately one-fourth of childhood T-cell ALL cases. However, TAL1 is aberrantly expressed in the leukemic cells of 60% of children and 45% of adults with T-cell ALL, implicating additional pathogenic mechanisms leading to TAL1 overexpression. One such recently described mechanism is an activating mutation of a noncoding gene-regulatory element, which is described later in this chapter. TAL1 acts as a master regulatory protein during early hematopoietic development and is required for the generation of all blood cell lineages. However, it does not seem to be required for the generation and function of hematopoietic stem cells (HSCs) during adult hematopoiesis. TAL1 is a bona fide T-cell ALL oncogene, as its aberrant expression in murine T-cell progenitors induces T-cell ALL.

TAL1 binds DNA in complex with other transcription factors including TCF3 (also known as E2A), HEB, LMO1/2, GATA3, RUNX1, and MYB. TAL1 binding can activate or repress the expression of its target genes, and a number of these targets have been implicated in T-cell transformation. In murine T-cell progenitors, TAL1 expression inhibits TCF3 transcriptional activity, and loss of TCF3 function induces T-cell leukemias in mice, supporting a role for TAL1-mediated inhibition of TCF3 function in leukemogenesis. The TAL1 complex binds and upregulates the expression of several of its own core components, including TAL1, GATA3, RUNX1, and MYB, thus forming a positive feedback loop that reinforces the activity of this oncogenic transcriptional complex. TAL1 also upregulates the expression of TRIB2, a gene whose overexpression in mouse bone marrow cells induces myeloid leukemia and whose expression is required for the survival of TAL1-overexpressing T-cell ALL. Furthermore, TAL1 also upregulates the expression of the microRNA mir223, which promotes leukemic cell survival by downregulating the expression of the FBXW7 tumor suppressor.

The LIM-only domain genes, LMO1 and LMO2, are also involved in recurrent chromosomal translocations in T-cell ALL. These genes encode transcription factors that interact with TAL1 in erythroid cells and in T-cell leukemias. Homozygous disruption of LMO2 in mice phenocopies the hematopoietic defect of TAL1 knock-outs, suggesting that these proteins function together during hematopoietic development. In addition, overexpression of LMO1 and LMO2 in murine thymocytes leads to T-cell transformation and accelerates the onset of leukemias in TAL1 transgenic mice.

Homeobox Genes

The homeobox gene TLX1 (also known as HOX11) is the founding member of a family of homeobox genes that includes TLX2 (HOX11L1) and TLX3 (HOX11L2), each of which plays key roles in embryonic development. TLX1 was originally isolated from the recurrent t(10;14) translocation in T-cell ALL and is aberrantly expressed in 5% of pediatric and approximately 25% of adult T-cell ALL cases. TLX3 is also involved in a recurrent t(5;14)(q35;q32) translocation and is overexpressed in approximately 25% of pediatric but in only 5% of adult T-cell ALL. TLX1 and TLX3 encode very similar proteins, suggesting they have similar oncogenic mechanisms. Overexpression of TLX1 or TLX3 induces differentiation arrest at a cortical stage of T-cell development, an effect that is mediated by TLX-induced transcriptional repression of the pre-TCR alpha, whose expression is required for progression beyond this stage in normal T-cell development. TLX1 expression in murine T-cell progenitors induces T-cell ALL, and these tumor cells have a defective mitotic checkpoint due to transcriptional repression of the checkpoint kinase CHEK1. These findings, together with the previous observation that TLX1 binds the catalytic subunits of the phosphatases PP2A and PP1 and disrupts the G2/M checkpoint, thus provide a mechanistic explanation for the association of TLX1/TLX3 overexpression with aneuploidy, which is otherwise rare in human T-cell ALL. RUNX1, which is directly bound and repressed by TLX1 and TLX3, has been implicated as a downstream mediator of the oncogenic function of these transcription factors in T-cell ALL.

The cluster of HOXA genes on chromosome 7 is affected by a recurrent chromosomal inversion that places it in the vicinity of TCR beta gene-regulatory elements, leading to aberrant expression of the entire HOXA cluster in approximately 5% of cases of T-cell ALL. Many of these cases also carry cooperating oncogenic lesions consisting of NOTCH1 gene mutations and deletions of 9p21. This translocation provides additional evidence for the role of aberrant HOXA activation in leukemogenesis and in the pathogenesis of KMT2A- and CALM-AF10 - rearranged leukemias, as reviewed in more detail in the following sections.

DUX4 Gene Rearrangements in B-Cell Acute Lymphoblastic Leukemia The double homeobox 4 (DUX4) gene encodes a homeobox-containing protein and is located on chromosome 4q within a sub-telomeric D4Z4 repeat region. In normal lymphoid cells, DUX4 undergoes epigenetic silencing. Recent genomic studies, however, have identified recurrent gene rearrangements in approximately four percent of B-cell ALL cases that fuse DUX4 to either IGH or, less commonly, ERG, a member of the erythroblast transformation-specific (ETS) family of transcription factors and an important regulator of hematopoietic cells. The juxtaposition of DUX4 to the regulatory region of IGH or ERG results in DUX4 overexpression in lymphoblasts. This overexpression leads to deregulation of ERG through binding of DUX4 to an alternative transcription initiation site within intron 6 of ERG, leading to truncated form of ERG that lacks the N-terminal pointed domain and central regulatory domains, but retains the DNA-binding ETS and transactivation domains, called ERGalt. Mice transplanted with ERGalt develop lymphoid malignancies, suggesting that expression of ERGalt directly induces lymphoid leukemogenesis. DUX4 gene rearrangements commonly co-occur with ERG deletions in B-cell ALL, suggesting that ERG deletions are secondary cooperating events in this subtype of B-cell ALL. Interestingly, IKZF1 deletions are commonly found in DUX4/ERG mutated B-cell ALL; however, unlike other ALL subtypes, IKZF1 deletions are not associated with a poor outcome in this group of patients.

ETV6-RUNX1 Fusions in B-Cell Acute Lymphoblastic Leukemia

Although most t(12;21) translocations are not detectable by standard cytogenetic analysis, this translocation is detectable by molecular techniques in approximately 25% of childhood B-cell ALL, making this the most common translocation in pediatric ALL (see Fig. 66.1 ). The ETV6–RUNX1 translocation often arises prenatally and is likely to be the initiating mutation in at least a subset of ALL, as evidenced by the identification of identical ETV6–RUNX1 translocations in identical twins with concordant ALL, and in retrospectively analyzed neonatal blood specimens of children who were diagnosed with ALL many years later. However, ETV6–RUNX1 alone is not sufficient for leukemogenesis because the incidence of detectable ETV6–RUNX1 fusions in the blood of normal newborns is about 100-fold greater than the incidence of leukemia.

The molecular mechanisms mediating ETV6–RUNX1 induced leukemogenesis remain poorly understood. This fusion gene encodes a chimeric protein that contains the helix–loop–helix (HLH) domain of ETV6 fused to nearly all of RUNX1 (also known as AML1 or CBFA2), including both the transactivation domain and the DNA- and protein-binding Runt homology domain. Both of these genes are found in other leukemia-related translocations, and both are essential for normal hematopoiesis. ETV6 was first identified in the t(5;12) in chronic myelomonocytic leukemia, where it is fused to the platelet-derived growth factor receptor gene (PDGFRB), and is also fused to ABL, MN1, and EVI1 in AML and to JAK2 in T-cell ALL. ETV6 is required for fetal hematopoiesis in the mouse. Interestingly, the inactivation of ETV6 in adult mice leads to the selective loss of HSCs from adult bone marrow, but hematopoiesis is sustained by committed precursors. Many ETV6–RUNX1 leukemias show the loss of the normal ETV6 allele, suggesting that the leukemogenic effect of ETV6-RUNX1 may be mediated in part by the loss of wildtype (WT) ETV6 function. Interestingly, germline loss-of-function ETV6 mutations have been identified in patients with familial thrombocytopenia and a predisposition to hematologic malignancies including ALL (as described in further detail later in this chapter), further implicating WT ETV6 as an ALL tumor suppressor.

RUNX1 is the DNA-binding component of the RUNX1–CBFß transcription factor complex disrupted by the t(8;21), t(3;21), and inv(16) in AML. RUNX1 is a transcription factor that is required for the expression of several hematopoietic genes involved in myeloid and lymphoid development, including PU.1 and IL-3, although it can also act as a transcriptional repressor in some settings. Homozygous disruption of the murine RUNX1 or CBFB genes results in the lack of definitive hematopoiesis, indicating that genes regulated by RUNX1 are essential for normal hematopoietic development. Additionally, rare familial mutations in the RUNX1 DNA-binding domain lead to the familial platelet disorder and predisposition to both myeloid and lymphoid malignancies.

The presence of the ETV6–RUNX1 translocation is associated with a favorable prognosis, with long-term event-free survival rates of approximately 90% in a number of studies.

TCF3-PBX1 Fusion Genes in B-Cell Acute Lymphoblastic Leukemia

Another example of an oncogenic chimeric transcription factor is the TCF3-PBX1 (previously known as E2A-PBX1) rearrangement, which results from the t(1;19)(q23;p13) chromosomal translocation present in about 5% of all B-lineage ALLs and in 25% of cases with a pre-B (cytoplasmic Ig-positive) phenotype. This translocation fuses the two N-terminal transactivation domains of the TCF3 transcription factor on chromosome 19 to the DNA-binding domain of the homeobox gene PBX1, leading to the expression of hybrid TCF3-PBX1 oncoproteins. The transforming potential of TCF3–PBX1 was first demonstrated by the rapid induction of AML in lethally irradiated mice repopulated with hematopoietic progenitors transduced with TCF3–PBX1 genes. This fusion has also been shown to transform NIH-3T3 fibroblasts and induce T-cell lymphomas in transgenic mice. Additional studies have shown that deletion of one of the TCF3 activation domains diminishes its transforming activity, but deletion of the PBX1 homeodomain has no effect. Historically, the presence of the TCF3–PBX1 translocation was associated with an inferior prognosis, but this no longer is the case with contemporary protocols for childhood ALL.

TCF3–HLF Fusion Genes in B-Cell Acute Lymphoblastic Leukemia

The t(17;19) is a rare recurrent chromosomal translocation that fuses the amino-terminal transactivation domains of TCF3 to the C-terminal DNA binding and dimerization domains of HLF, a basic leucine zipper domain transcription factor. Although TCF3–HLF can bind DNA either as a homodimer or as a heterodimer with HLF and related proteins, no other PAR proteins are expressed in hematopoietic cells, and the TCF3–HLF fusion binds DNA as a homodimer in cells harboring the t(17;19). Similar to TCF3–PBX1, TCF3–HLF can transform NIH–3T3 fibroblasts, a process that requires the HLF leucine zipper domain and the TCF3 transactivation domains, and can induce lymphoid tumors in transgenic mice. Recent transcriptomic analysis has revealed that the TCF3–HLF fusion leads to a gene expression signature enriched for stem cell and myeloid markers, suggesting that this fusion leads to cellular reprogramming of a lymphoid progenitor to a more primitive state. Additionally, another major consequence of TCF3–HLF expression in lymphoid precursors is the inhibition of apoptotic cell death. In normal pro-B lymphocytes, the expression of TCF3–HLF blocks apoptosis induction by either IL-3 or p53. In TCF3-HLF expressing human ALL cells, the inhibition of TCF3–HLF function by expression of a dominant-negative form of TCF3–HLF results in apoptosis induction. HLF is the mammalian homolog of the worm protein ces-2, a transcription factor that is necessary for the death of two specific nerve cells during Caenorhabditis elegans development. This pathway, which is evolutionarily conserved, is inhibited by the TCF3-HLF fusion. Thus, in contrast to the proapoptotic role of the WT HLF homolog (ces-2) in worms, TCF3–HLF blocks apoptosis by inducing the expression of SLUG, a transcription factor that blocks DNA damage-induced apoptosis in hematopoietic cells. The t(17;19) occurs in less than 1% of ALL cases, is associated with characteristic clinical features including adolescent age, disseminated intravascular coagulation and hypercalcemia at diagnosis, as well as a high risk of treatment failure leading to a dismal prognosis.

MEF2D Gene Fusions in B-Cell Acute Lymphoblastic Leukemia

Cryptic fusions involving MEF2D at chromosome 1q21–22 have recently been identified through RNA sequencing studies. MEF2D, located at chromosome 1q21–22, encodes myocyte enhancer factor 2D and is a member of the myocyte enhancer factor family of transcription factors that are expressed throughout normal B-cell development. Previous studies have shown that the inactivation of Mef2d results in maturation arrest at the pre-B-cell stage of lymphoid development. In these cases, the amino terminus of MEF2D fuses in frame with the carboxy terminus of the fusion partner. Several fusion partners of MEF2D have been identified, including most commonly BCL9, which is a component of the WNT/β-catenin signaling cascade. The prognostic implication of MEF2D gene fusions has not yet been established.

ZNF384 Gene Fusions in B-Cell Acute Lymphoblastic Leukemia

Fusions involving the zinc-finger protein 384 (ZNF384) gene, which encodes a transcription factor that regulates promoters of the extracellular matrix, have recently been identified through RNA sequencing and transcriptomic analyses in B-cell ALL. To date, eight fusion partners have been identified, including BMP2K, TCF3, TAF15, EWSR, EP300, CREBBP, SYNRG, and ARID1B. ZNF384-related fusion genes have demonstrated the transformation of NIH3T3 fibroblasts in vitro; however, their exact cellular consequences and role in leukemogenesis remain unknown. B-cell ALL cases that harbor ZNF384-related fusions are characterized by a unique immunophenotype that is weak or negative for CD10 and positive for the myeloid markers, CD13 and CD33, but are variable in clinical presentation. ZNF384 gene fusions have also been observed in up to 50% of B-cell/myeloid cases of MPAL.

CALM–AF10 Fusion Gene in T-Cell Acute Lymphoblastic Leukemia

The t(10;11)(p13;q14) is detected in approximately 3% to 10% of T-cell ALL cases and in occasional AML cases. This translocation results in the fusion of CALM (also known as PICALM), encoding a protein with high homology to the murine clathrin assembly protein ap3, with AF10, a gene identified as a KMT2A partner in the KMT2A–AF10 fusion resulting from the t(10;11)(p13;q23). The expression of the CALM-AF10 fusion transcript has been associated with early arrest in T-cell development and with differentiation into the gamma-delta lineage in T-cell ALL. Additionally, aberrant upregulation of HOX gene expression appears to be involved in CALM–AF10-mediated leukemogenesis, at least in acute myeloid leukemia cells that carry this translocation. Interestingly, analysis of a mouse model of CALM–AF10-induced acute myeloid leukemia suggests that the leukemic stem cell in this model has lymphoid characteristics, and cells from human patients with AML can be identified that have similar characteristics to the disease-propagating cell in this animal model.

SPI1 Fusion Genes in T-Cell Acute Lymphoblastic Leukemia

Sequencing studies pf pediatric T-cell ALL have recently identified recurrent SPI1 fusions (STMN1-SPI1 and TCF7-SPI1) in approximately 4% of cases and are associated with poor outcome. SPI1 is a member of the ETS family of transcription factors and is expressed in hematopoietic cells, where its tight regulation is critical for normal HSC development. During normal T-cell development, SPI1 is expressed in prethymic and early T-cell progenitors and turned off during later stages of T-cell development. The fusion places SPI1 under the control of TCF7 or STMN1 promoter and results in the overexpression of SPI1, regardless of fusion partner. In vitro studies demonstrate that expression of SPI1 fusion genes results in cell proliferation and differentiation arrest during the double-negative (DN) stage of T-cell development, thereby likely contributing to T-cell leukemogenesis.

NOTCH1 in T-Cell Acute Lymphoblastic Leukemia

The NOTCH1 gene was originally discovered as a partner gene in an exceedingly rare t(7;9) chromosomal translocation in T-cell ALL, in which NOTCH1 is truncated and placed under the control of the TCRβ locus. Despite the rarity of NOTCH1 translocations, a search for point mutations within the NOTCH1 gene revealed activating mutations in more than 50% of cases of T-cell ALL. NOTCH1 plays several critical roles during T-cell development, and its overexpression in murine hematopoietic cells potently drives T-cell ALL. NOTCH1 is a transmembrane protein that is proteolytically processed during its transit to the cell surface, where it exists as a heterodimer consisting of extracellular and transmembrane subunits. Upon ligand binding, the transmembrane subunit undergoes additional proteolytic cleavage within the plasma membrane, leading to the release of its intracellular domain, known as ICN1 (intracellular domain of NOTCH1), into the cytosol. ICN1 subsequently translocates into the nucleus, where it is active as a transcription factor. Activating NOTCH1 mutations in T-cell ALL can occur as either missense mutations in the heterodimerization domain, allowing constitutive proteolytic activation of the ICN1 domain, or as frameshift or stop codon mutations that lead to truncation of the PEST domain. The PEST domain regulates proteasomal degradation of the protein, and these PEST-inactivating mutations result in aberrant stabilization of ICN1 protein. Mutations in both regions are often found on the same allele in cases of T-cell ALL and are synergistic in increasing NOTCH1 transcriptional output. Additionally, the NOTCH1 oncoprotein can also be stabilized by inactivating mutations of tumor suppressor genes involved in its proteasomal degradation, including FBXW7 and Cyclin C, as discussed later in this chapter. MYC is an important transcriptional target of NOTCH1, and it mediates many of the leukemogenic properties of NOTCH1 in human T-cell ALL cells. However, NOTCH1 also has MYC-independent oncogenic activity, as evidenced by its ability to induce T-cell ALL in zebrafish, where NOTCH1 does not upregulate MYC expression. Additional NOTCH1 targets implicated in T-cell ALL pathogenesis include the IL7R interleukin receptor and the long noncoding RNA LUNAR1, which is upregulated by NOTCH1 and functions to upregulate expression of the growth factor receptor IGF1R.

Small-molecule inhibitors of γ-secretase impair the proteolytic activation of NOTCH, but early clinical trial results with these agents in relapsed/refractory T-cell ALL have been disappointing. Investigation of mechanisms of resistance to NOTCH1 inhibition has revealed several potential strategies for therapeutic intervention. Activation of the phosphoinositide-3-kinase (PI3K)-AKT pathway, which commonly results from loss of its negative regulator PTEN in T-cell ALL, provides one mechanism for resistance to NOTCH1 inhibition. The PTEN-PI3K-AKT pathway also mediates resistance to MYC inhibition in this disease. However, work in a murine model of Kras-induced T-ALL, where leukemias commonly acquire activating NOTCH1 mutations and are sensitive to PI3K inhibitors, revealed that the development of resistance to PI3K inhibition was unexpectedly associated with the loss of oncogenic NOTCH1 signaling. This finding thus raises the possibility that dual PI3K-NOTCH1 inhibition may actually promote the emergence of drug resistance. Additional studies are needed to define the optimal strategy for clinical application of NOTCH1 and PI3K inhibition in patients with T-cell ALL.

Recent work has also revealed that some T-cell ALL cases in which the bulk cell population is sensitive to NOTCH1 inhibition harbor minor populations of so-called “persister” cells that are resistant to NOTCH1 inhibitors. This “persister” state is not driven by genetic mutations but is instead a reversible cellular state characterized by chromatin compaction. These NOTCH1 inhibitor-resistant cells maintain the expression of MYC, a crucial downstream target of NOTCH1, despite effective inhibition of NOTCH1 activity. However, the survival of these cells is specifically dependent on BRD4, a transcriptional regulator whose ability to bind acetylated chromatin can be specifically inhibited using small molecules. Combination therapy with inhibitors of both BRD4 and NOTCH1 can block the emergence of this resistance mechanism, and this approach has promising in vivo activity in preclinical models.