mutations that targeted genes in key cellular pathways in B-ALL are identified and are
applied to characterize new subtypes or increase the new insights into known ALL
subtypes, such as BCR/ABL1-like ALL (~15%) (9, 10), intrachromosomal amplification
of chromosome 21 (iAMP21, ~2%) (11, 12), IGH- and CRLF2-rearrangement (5-7%)
(13), ERG-deregulated ALL (~7%) (14), PAX5-deletion (3%) /mutation (5-7%) /
rearrangement (2-3%) (5), and IKZF1-deregulated ALL (~15%) (9, 15).
1.3. ETV6/RUNX1-positive B-ALL
1.3.1. ETV6/RUNX1 fusion gene
The t(12;21) (p13;q22) translocation, which results in ETV6/RUNX1 fusion gene,
was first reported by two different group in 1995 (16, 17). It is the most common
chromosomal rearrangement in childhood B-ALL cases but less prevalent in adult
patients (1, 18). The incidence of t(12;21) in B-ALL is approximately 15-25%, and
patients with this translocation usually have excellent outcome (19). This rearrangement
is not able to be detected by conventional cytogenetic analysis but is readily detected by
fluorescent in situ hybridization and molecular techniques, such as RT-PCR and
quantitative RT-PCR (20-22).
The ETV6 gene, which is very large (240 kb) and consists of eight exons coding for
an ETS-like putative transcription factor containing a helix-loop-helix (HLH) and a
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DNA-binding domain is on human chromosome 12p13 (23). The RUNX1 gene, which
belongs to the runt domain gene family of transcription factors, is on human
chromosome 21q22 and spans 260 kb consisting of 12 exons (24). Both ETV6 and
RUNX1 genes demonstrated critical roles in hematopoiesis in knockout mice studies
(25-27), and they are also frequently targeted by rearrangements and mutations in
leukemia (28, 29).
The breakpoints on ETV6 gene are clustered in a 15 kb region between exon 5 and
6 (23), and the breakpoints on RUNX1 gene may occur either in the ~100 kb intron 1 or
intron 2 (Appendix IIA) (18, 30). Most of all, the ETV6/RUNX1 fusion transcript shows
a joining of exon 5 of ETV6 to the second exon of RUNX1, while the junction occurred
at the third exon of RUNX1 is less frequently seen (Appendix IIB) (30). Wherever the
breakpoints occurred, these all result in fusion of the 5ʹ portion of ETV6 and almost the
entire coding region of RUNX1 gene.
1.3.2. Structure and function of ETV6/RUNX1 fusion protein
The ETV6/RUNX1 fusion protein is composed of the N-terminal
non-DNA-binding region of ETV6 and nearly complete RUNX1 protein, including the
DNA-binding and activation region, which is responsible for the essential function of
the fusion protein (Appendix III) (16, 18, 31). The ETV6 protein acts as a DNA-binding
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transcriptional repressor, while RUNX1 can be a DNA-binding transcriptional activator
or repressor depending on the promoter specificity or cell context (18). In contrast to
RUNX1, transiently expressed ETV6/RUNX1 fusion protein generally represses the activities of reporter constructs driven by regulatory regions derived from
hematopoietic-specific genes (18). The ETV6/RUNX1 fusion protein acts as an aberrant
transcription factor that can interfere with the normal functions of wild-type ETV6 and
RUNX1 through multiple mechanisms. ETV6/RUNX1 can dimerize with wild-type
ETV6 through the HLH domain and disrupt the activity of ETV6 (31, 32).
ETV6/RUNX1 may also bind to RUNX1 target DNA sequences and recruit
transcriptional corepressors including mSinA, N-coR, and histone deacetylase-3
(HDAC3) via the fusion part of ETV6, resulting in dysregulated RUNX1-dependent
transcription (Appendix IV) (31, 33, 34).
1.3.3. Role of ETV6/RUNX1 in leukemogenesis of B-ALL
Analysis of monozygotic twins with concordant leukemia and retrospective
screening of neonatal blood spots of patients with leukemia indicate that chromosomal
translocations characteristic of pediatric leukemia often arise prenatally (35). However,
in normal cord blood ETV6/RUNX1 is detected at a frequency that is 100-fold greater
than the risk of the corresponding leukemia (36). Mouse models demonstrate that
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expression of ETV6/RUNX1 in murine bone morrow stem cells impedes B cell
differentiation from the earliest stages of B cell development with a particular marked impact at the transition from pro-B to pre-B cell stages. Although the accumulation of
both multipotent and B-cell progenitors in vivo, ETV6/RUNX1 is insufficient to induce
leukemia by itself (37, 38). Collectively, these data suggests that ETV6/RUNX1 is a
frequent prenatal first hit in childhood leukemia which can initiate a preleukemic
phenotype remaining covert for up to 15 years but is insufficient for clinical leukemia.
1.4. MicroRNAs
1.4.1. Overview
MicroRNAs (miRNAs) are a group of single-stranded, endogenously initiated
non-coding RNAs which are first discovered in the early 1990s (39). They are
transcribed by RNA polymerase II as part of capped and polyadenylated primary
transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary
transcript is cleaved by the Drosha ribonuclease III enzyme to produce an
approximately 70 to 100- nucleotide stem-loop precursor miRNA (pre-miRNA), which
is exported to cytoplasm and is further cleaved by the Dicer ribonuclease to generate the 20-23 nucleotides mature miRNA products from 3ʹ and/or 5ʹ arms (40). When two
mature miRNAs originate from opposite arms of the same pre-miRNA and express
17
similar amounts, they are represented as -3p and -5p (41). In human cells, the mature
miRNAs incorporate into a RNA-induced silencing complex (RISC) and then target
mRNAs for degradation or translational repression via partial or perfect
complementarity to the mRNA 3' untranslated region (3' UTR) through specific seed
sequences (Appendix V) (40, 42). By this way, miRNAs can downregulate gene
expression at the post-transcriptional level. In the recent years, there has been increasing
evidence that miRNAs also bind in the coding region to inhibit translation (43),
implying that miRNAs may flexibly tune the time-scale and magnitude of
post-transcriptional regulation through combination of multiple ways (44). Thus,
miRNAs have the ability to control fundamental cellular functions such as survival,
differentiation, apoptosis, and cell cycle.
1.4.2. MicroRNAs in hematopoiesis and leukemogenesis
A variety of miRNAs have been identified as important regulators of
hematopoiesis (45). For instance, miR-221/222 inhibits the erythropoiesis (46),
miR-223 is essential to modulate myeloid differentiation (47), miR-181a and miR-150
are dynamically regulated during T-cell and B-cell development, respectively, and
premature expression of certain miRNAs in hematopoietic progenitors may impair T-
and B-cell development at the stage transition during maturation (48-50).
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MiRNA expression in hematological malignancies has also been extensively
studied. Some information about factors that modulate miRNA expression is now
available. Dysregulation of miRNA expression is frequently associated with cytogenetic
abnormalities, and indeed certain abnormalities have direct impacts on aberrant
miRNAs expressions (45). RUNX1/ETO, the most common acute myeloid leukemia–
associated fusion protein resulting from t(8;21), was first reported to directly repress
miR-223 expression by triggering chromatin remodeling and epigenetic silencing,
which in turn blocks the differentiation of myeloid precursor cells (51).
1.4.3. MIR181A1 gene
MIR181A1 gene is located on human chromosome 1q32.1 and is only 62 bp distant
from MIR181B1 gene, they are considered sharing the same primary transcript. There is
only one exon in MIR181A1 gene and expressed a pre-miRNA with 110 bp in length
referred to hsa-mir-181a-1. Hsa-mir-181a-1 will further generate two mature miRNAs
including miR-181a-3p and -5p, which are referred to miR-181a-1 and miR-181a,
respectively (Figure 1A). MIR181A1 belongs to the miR-181 family which consists of
mir-181a/b1, mir-181a/b2 and mir-181c/d, producing four highly similar mature 5p
miRNAs (miR-181a, miR-181b, miR-181c and miR-181d, respectively) with identical
seed sequence and a slight difference in 1~4 bp from three polycistronic transcripts
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(Figure 1B).
Although both 3p and 5p of hsa-mir-181a can be detected in tissues, previous
reports only demonstrated that miR-181a targets various mRNAs and has physiological
roles and pathological meanings (52-54), while the function of miR-181a-1 remains
unclear. In healthy cells, miR-181a regulates B-cell development, influences T-cell
sensitivity to antigens by modulating T-cell receptor signaling, and is involved in early
steps of hematopoiesis (55). A tumor suppressor activity of miR-181a is reported in
chronic lymphocytic leukemia (CLL) (56, 57), glioma (53), and astrocytoma (54). In
addition, ectopic expression of miR-181a has been shown to sensitizes acute myeloid
leukemia (AML) cell lines to chemotherapy (58), and enhance the effect of radiation
treatment on malignant glioma cells via down-regulation of the Bcl-2 protein (59).
1.4.4. MicroRNAs associated with ETV6/RUNX1
Recent studies have shown that aberrant miRNA expression also plays an
important role in malignant transformation of ETV6/RUNX1 ALL. A highly expressed
miR-125b-2 cluster was found in ETV6/RUNX1 ALL and may provide leukemic cells
with a survival advantage against growth inhibitory signals in a p53-independent
mechanism (60). In addition, ETV6/RUNX1 was shown to regulate the cellular level of
the Survivin protein and apoptosis via suppression of miR-494 and miR-320a
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expression by direct binding to the promoter regions of their encoding genes (61).