Molecular pathogenesis of secondary acute promyelocytic leukemia.

Abstract

Balanced chromosomal translocations that generate chimeric oncoproteins are considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent is the t(15;17)(q22;q21), which fuses the PML and RARA genes, giving rise to acute promyelocytic leukemia (APL). An increasing proportion of APL cases are therapy-related (t-APL), which develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA topoisomerase II (topoII), particularly mitoxantrone and epirubicin. To gain insights into molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation breakpoints in a series of t-APLs, which revealed significant clustering according to the nature of the drug exposure. Remarkably, in approximately half of t-APL cases arising following mitoxantrone treatment for breast cancer or multiple sclerosis, the chromosome 15 breakpoint fell within an 8-bp "hotspot" region in PML intron 6, which was confirmed to be a preferential site of topoII-mediated DNA cleavage induced by mitoxantrone. Chromosome 15 breakpoints falling outside the "hotspot", and the corresponding RARA breakpoints were also shown to be functional topoII cleavage sites. The observation that particular regions of the PML and RARA loci are susceptible to topoII-mediated DNA damage induced by epirubicin and mitoxantrone may underlie the propensity of these agents to cause APL.

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Mediterr J Hematol Infect Dis 2011; 3; Open Journal System
MEDITERRANEAN JOURNAL OF HEMATOLOGY AND INFECTIOUS DISEASES MEDITERRANEAN JOURNAL OF HEMATOLOGY AND INFECTIOUS DISEASES
www.mjhid.org ISSN 2035-3006
Review Articles
Molecular Pathogenesis of Secondary Acute
M. Joannides1, A. N. Mays1, A. R. Mistry
Osheroff6, E. Solomon1and D. Grimwade
1Department of Medical & Molecular Genetics, King’s College London School
2Department of Biopathology & Fondazione Santa Lucia, University of Tor Vergata, Rome, Italy.
3III.Medizinische Klinik,Universitätsmedizin Mannheim, Mannheim, Germany.
4Department of Epidemiology and Biostatistics, University of California
5Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, USA.
6The Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, USA. The Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, USA.The Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nashville, USA.
Correspondence to: David Grimwade,
Tower Wing, Guy’s Hospital, London SE1 9RT, UK. Tel: +44 207 188 3699,
david.grimwade@genetics.kcl.ac.uk
Competing interests: The authors have d
Published: October 24, 2011
Received: August 25, 2011
Accepted: September 20, 2011
Mediterr J Hematol Infect Dis 2011, 3(1): e20110
This article is available from: http://www.mjhid.org/article/view/9085
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0
provided the original work is properly cited.provided the original work is properly cited.
Abstract: Balanced chromosomal translocations that generate chimeric oncoproteins are
considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent
is the t(15;17)(q22;q21), which fuses the
leukemia (APL). An increasing proportion of APL cases are therapy
develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA
topoisomerase II (topoII), particu
molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation
breakpoints in a series of t-APLs, which revealed significant clustering according
the drug exposure. Remarkably, in approximately half of t
mitoxantrone treatment for breast
within an 8-bp “hotspot” region in
topoII-mediated DNA cleavage induced by mitoxantrone.
outside the “hotspot”, and the corresponding
topoII cleavage sites. The observation that particular r
susceptible to topoII-mediated DNA damage induced by epirubicin and mitoxantrone may underlie
the propensity of these agents to cause APL.the propensity of these agents to cause APL.
Introduction. Acute myeloid leukemia (AML) is
characterized by a spectrum of recurring chromosomal characterized by a spectrum of recurring chromosomal
; Open Journal System
f Secondary Acute Promyelocytic Leukemia Promyelocytic Leukemia.
, A. R. Mistry1, S. K. Hasan2, A. Reiter3, J. L. Wiemels4, C. A. Felix
and D. Grimwade1
Department of Medical & Molecular Genetics, King’s College London School of Medicine, UK.
Department of Biopathology & Fondazione Santa Lucia, University of Tor Vergata, Rome, Italy.
III.Medizinische Klinik,Universitätsmedizin Mannheim, Mannheim, Germany.
Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, USA.
Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, USA.
Cancer Genetics Lab, Dept. of Medical & Molecular Genetics, 8
, Guy’s Hospital, London SE1 9RT, UK. Tel: +44 207 188 3699, Fax: +44 207 188 2585. E
have declared that no competing interests exist.
: e2011045, DOI 10.4084/MJHID.2011.045
http://www.mjhid.org/article/view/9085
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, ), which permits unrestricted use, distribution, and reproduction in any medium,
osomal translocations that generate chimeric oncoproteins are
considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent
is the t(15;17)(q22;q21), which fuses the PML and RARA genes, giving rise to acute promyelocytic
leukemia (APL). An increasing proportion of APL cases are therapy-related (t
develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA
topoisomerase II (topoII), particularly mitoxantrone and epirubicin. To gain insights into
molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation
APLs, which revealed significant clustering according
exposure. Remarkably, in approximately half of t-APL cases arising following
mitoxantrone treatment for breast cancer or multiple sclerosis, the chromosome 15 breakpoint fell
bp “hotspot” region in PML intron 6, which was confirmed to be a pref
mediated DNA cleavage induced by mitoxantrone. Chromosome 15 breakpoints falling
outside the “hotspot”, and the corresponding RARA breakpoints were also shown to be functional
The observation that particular regions of the PML
mediated DNA damage induced by epirubicin and mitoxantrone may underlie mediated DNA damage induced by epirubicin and mitoxantrone may underlie
cute myeloid leukemia (AML) is abnormalities, which distinguish biologically and
prognostically distinct subtypes of disease (reviewed prognostically distinct subtypes of disease (reviewed 1).
MEDITERRANEAN JOURNAL OF HEMATOLOGY AND INFECTIOUS DISEASES
, C. A. Felix5, F. Lo Coco2, N.
of Medicine, UK.
Department of Biopathology & Fondazione Santa Lucia, University of Tor Vergata, Rome, Italy.
, San Francisco, San Francisco, USA.
Cancer Genetics Lab, Dept. of Medical & Molecular Genetics, 8thFloor,
Fax: +44 207 188 2585. E-mail:
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
osomal translocations that generate chimeric oncoproteins are
considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent
genes, giving rise to acute promyelocytic
related (t-APL), which
develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA
larly mitoxantrone and epirubicin. To gain insights into
molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation
APLs, which revealed significant clustering according to the nature of
APL cases arising following
or multiple sclerosis, the chromosome 15 breakpoint fell
intron 6, which was confirmed to be a preferential site of
Chromosome 15 breakpoints falling
breakpoints were also shown to be functional
PML and RARA loci are
abnormalities, which distinguish biologically and

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Mediterr J Hematol Infect Dis 2011; 3: Open Journal System
To date, more than one hundred balanced chromosomal
rearrangements (translocations,
inversions) have been identified and cloned,2with
evidence suggesting that these are critical initiating
events in the pathogenesis of AML. Identification of
the genes involved in chromosomal rearrangements has
provided major insights into the regulation of normal
hematopoiesis and how disruption of key transcription
factors and epigenetic modulators promote leukemic
transformation. A number of genes, including MLL at
11q23, NUP98 at 11p15, RARA at 17q21 and RUNX1
at 21q22, are recurrent targets of chromosomal
rearrangements in AML, being fused to a range of
potential partner genes (reviewed
chromosomal rearrangements
particular loci also have been noted as a feature of
secondary acute leukemias arising as a complication of
prior therapy involving drugs targeting topoisomerase
II (topoII), which are widely used in the treatment of a
variety of tumors.4-13TopoII is a critical enzyme that
relaxes supercoiled DNA and removes knots and
tangles from the genome by transiently cleaving and
religating both strands of the double helix via the
formation of a covalent cleavage intermediate
(reviewed
anthracyclines (e.g. epirubicin) and anthracenediones
(e.g. mitoxantrone) act as topoII poisons, inducing
DNA damage by disrupting the cleavage-religation
equilibrium and increasing the concentration of DNA
topoII covalent complexes, which leads to apoptosis of
the tumor cells.14
The association between
chemotherapeutic agents
development of leukemias with balanced chromosomal
rearrangements has naturally implicated the enzyme in
this process, but the mechanisms involved have
remained subject to debate. One hypothesis takes into
account reports that leukemia-associated translocations
can be detected in hematopoietic cells derived from
healthy individuals without overt leukemia,15,16
suggesting that administration of chemotherapy and/or
radiotherapy provides a selective advantage to
progenitors with pre-existing translocations during
regrowth of depopulated bone marrow. Moreover, the
exposure to DNA damaging agents could lead to the
acquisition of additional mutations that cooperate with
the chimeric fusion protein generated by the
translocation to induce leukemic transformation. A
second hypothesis, based on findings with transformed
cells, raised the possibility that agents targeting topoII
could lead to the formation of chromosomal
translocations through an indirect mechanism involving
induction of apoptotic nucleases.17-20Interestingly, Rolf
Marschalek and colleagues provide evidence for a third
potential mechanism, showing that the region of the
insertions and
3). Interestingly,
involving these
14). Epipodophyllotoxins (e.g. etoposide),
exposure
topoII
to
targeting and
MLL locus where breakpoints associated with infant
and therapy-related leukemias cluster, colocalize with
an internal promoter element, highlighting the
relevance of chromatin structure and DNA topology in
the genesis of chromosomal translocations.21Finally,
the fourth hypothesis, which is based on increasing
biochemical and genetic evidence, suggests that in the
presence of a topo II-targeting chemotherapeutic agent,
topoII plays a direct role in generating double-stranded
DNA breaks in regions of the genome that are
particularly susceptible due to the nature of the
surrounding chromatin. Following aberrant repair,
these breaks go on to generate leukemia-associated
chromosomal translocations (reviewed 22).
Intriguingly, the nature of the drug exposure has a
bearing on the molecular phenotype of the resultant
secondary leukemia, with translocations involving the
MLL gene at 11q23 being particularly associated with
etoposide exposure.10,23,24Development of therapy-
related acute promyelocytic leukemia
characterized by the t(15;17)(q22;q21), has been linked
to treatment with mitoxantrone and epirubicin.12,25,26
The t(15;17) leads to fusion of the gene encoding the
myeloid transcription factor RAR (Retinoic Acid
Receptor Alpha) at 17q21 with a gene that was
previously unknown -
ProMyelocytic Leukemia), which has subsequently
been found to be involved in growth suppression and
regulation of apoptosis (reviewed 27). This subtype of
leukemia is of particular interest, being the first in
which molecularly targeted therapies (i.e., all-
transretinoic acid [ATRA] and arsenic trioxide [ATO])
have been successfully used in clinical practice.27
These agents act by inducing degradation of the PML-
RAR oncoprotein, leading to clinical remission and
have resulted in dramatic improvements in clinical
outcome (reviewed 28). They also offer a potentially
curative approach in patients with t-APL who have
already received significant doses of chemotherapy for
their previous condition and may be close to the
anthracycline ceiling, or who are considered unfit for
conventional therapy.29
The majority of t-APL cases arise in patients who
have undergone treatment for breast cancer, where
mitoxantrone and epirubicin have been widely
used.12,25,26,30In this setting, cumulative doses of
epirubicin of ≤720mg/m2have been associated with a
secondary leukemia risk of 0.37% at 8 years.31As more
patients survive their primary cancers, secondary
leukemias are becoming an increasing healthcare
problem.32
Although t-APL
uncommon, two case series have suggested that the
incidence has risen in recent years, with up to 20% of
APL patients presenting with secondary disease.25,30
(t-APL),
designated
PML(for
remains relatively

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Mediterr J Hematol Infect Dis 2011; 3: Open Journal System
Figure 1. Distribution of translocation breakpoints within the PML and RARA loci in t-APL cases arising following epirubicin and
mitoxantrone. PML exons are represented by red boxes, RARA exons in blue and introns are represented by black lines. Arrows indicate the
location of PML and RARA translocation breakpoints identified by long-range PCR and sequence analysis in patients with t-APL arising
following mitoxantrone (red arrows) or epirubicin (green arrows). In 12 patients mitoxantrone was used for treatment of multiple sclerosis
(MS). In the remaining 5 patients with mitoxantrone-related APL and the 6 patients with t-APL following epirubicin, these agents were used
for prior breast cancer. Significant breakpoint clustering was observed, with a “hotspot” identified in PML intron 6 (position 1482-9) in
mitoxantrone-related APL (following use of the drug for MS or breast cancer) and separate clusters associated with APL arising following
epirubicin exposure. Chromosomal breakpoints were confirmed to be preferential sites of drug-induced topoisomerase II cleavage in
functional assays (see Figure 2). Adapted from Mays et al.42with permission.
Investigation
Mitoxantrone-Related t-APL. As a first step to gain
insights into mechanisms underlying formation of the
t(15;17) we used long-range polymerase chain reaction
(PCR) and sequence analysis to define chromosomal
breakpoint locations, comparing the pattern between
patients presenting de novo (n=35) and those with t-
APL occurring following exposure to mitoxantrone
(n=6) or other agents/radiation therapy (n=7).33
Analysis of diagnostic samples from large cohorts of
patients with de novo APL has established that the
majority of chromosome 15 breakpoints fall within 3
breakpoint cluster regions (bcr) i.e. in intron 3 (bcr3),
intron 6 (bcr1) and exon 6 (bcr2) of the PML locus,
accounting for approximately 40%, 55% and 5% of
cases respectively.34Chromosome 17 breakpoints fall
within the ~17kb intron 2 of the RARA locus, such that
the PML-RAR fusion retains key functional domains
mediating DNA binding and interaction with
coactivator/corepressors,
ligand (i.e. retinoic acid).27While breakpoints observed
in de novoAPL appeared broadly distributed,
chromosome 15 breakpoints of each of the
mitoxantrone-related t-APLs fell within PML intron 6,
with 4 cases clustering within an 8-bp region (position
of Molecular Mechanisms in
retinoid-X-receptor and
1482-9)(see Figure 1).33Given that this intron is over
1kb in length, this clustering of breakpoints was highly
unlikely to have occurred by chance (p<0.001 by scan
statistics). To investigate this further, we used a
functional in vitro assay, in which substrates containing
the normal homologues of the PML and RARA
translocation breakpoints are 5’-end-labelled and
exposed to clinically relevant topoII poisons (e.g.
mitoxantrone, epirubicin, etoposide) in the absence or
presence of human topoII alpha; cleavage complexes
are trapped and cleavage sites mapped in relation to the
observed translocation breakpoints at the sequence
level.35-37These experiments demonstrated that the
breakpoint “hotspot,” identified in t-APL patients
previously treated with mitoxantrone for breast cancer,
corresponded precisely to a preferential mitoxantrone-
induced topoII-dependent DNA cleavage site at
position 1484 (see Figure 2).33
observed translocation breakpoint within the RARA
locus on chromosome 17 was confirmed to be a
preferred site of topoII-mediated DNA damage induced
by mitoxantrone (Figure 2).33
These data strongly implicated mitoxantrone in the
etiology of t-APL. However, it is important to consider
that the study of patients developing leukemia
Moreover, each

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Mediterr J Hematol Infect Dis 2011; 3: Open Journal System
Figure 2. Demonstration of mitoxantrone-induced topoisomerase II dependent DNA cleavage at translocation breakpoints in therapy-related
APL. A) In vitro DNA topoisomerase II (topoII) cleavage assay carried out for a PML substrate containing the breakpoints of 4 treatment-
related APL (t-APL) cases (F-8,-24,-25,-27) within the 8-bp breakpoint “hotspot” (positions 1482-1489). Patients received combination
chemotherapy including the topoII poison mitoxantrone for breast cancer. Control reactions were carried out in the absence of topoII (lanes
1-4), and in the presence of etoposide (VP16), etoposide catechol (VP16-OH), etoposide quinone (VP16-Q) and mitoxantrone (Mit).
Dideoxy sequencing reactions of the substrate are shown in lanes 5-8. Cleavage reactions were carried out by exposure to human topoII in
the absence of drug (lane 9), and in the presence of etoposide (lane 10), etoposide catechol (lane 11), etoposide quinone (lane 12) and
mitoxantrone (lane 13). Additional cleavage reactions were carried out to evaluate the heat-stability of cleavage complexes formed by
incubation at 75C for 10 min (lanes 14-18). The nucleotide shown by the dash is the 5’side of the cleavage site (-1 position), which
corresponds to the der(15) and der(17) translocation breakpoints in 4 cases of mitoxantrone-related APL (far right). The cleavage site at
position 1484 was observed in the absence of drug, and in the presence of etoposide, both etoposide metabolites and mitoxantrone (lanes 9-
13). Cleavage at this position was the strongest site observed in the presence of mitoxantrone (lane 13). Furthermore, the complexes formed
at this site were shown to be heat-stable in the presence of mitoxantrone (lane 18). Interestingly, a cleavage site at position 1502 is also
observed, which corresponds to a breakpoint detected in a case of de novo APL.
B) TopoII cleavage assay of normal homologue of der(15) and der(17) RARA translocation breakpoints in APL of one of the mitoxantrone-
related cases (F-8). The substrate spanning positions 2603 to 2871 of RARA intron 2 contained the translocation breakpoints. Dash at right
shows (-1) position of cleavage site corresponding to der(15) and der(17) translocation breakpoints (arrow far right). Adapted from Mistry et
al.33with permission.
following cancer therapy presents a challenge, given
that they have often been exposed to multiple cytotoxic
drugs in addition to radiotherapy. This makes it
difficult to identify the causative agent with any
certainty. Moreover, such patient populations could
feasibly be enriched for individuals at particular risk of
leukemia, having already developed one form of
cancer. Therefore, to provide further insights into
molecular mechanisms in topoII-related leukemias, we
analyzed a cohort of 12 patients collected from across
Europe who developed APL following the use of single
agent mitoxantrone to treat a benign condition,
multiple sclerosis (MS), and in whom there was no
history of previous cancer.38
breakpoints again were found to cluster at position
1484 within PML intron 6. Furthermore, shared
chromosome 17 breakpoints that were preferential sites
of mitoxantrone-induced topoII cleavage in functional
assays were identified within RARAintron 2 (Figure
Chromosome 15
1).38The series of mitoxantrone-related t-APL cases
analyzed has been further extended recently, with the
chromosome 15 breakpoint found to fall within the
“hotspot” region in 12 of 23 cases (52%).33,38,39
Comparison of the genomic breakpoint junction
regions with the native
translocations in mitoxantrone-related t-APL were
reciprocal, generally without loss or gain of
nucleotides.33,38
Presence of sequence homologies
between PML and RARA suggests that topoII-mediated
DNA damage may be repaired by the canonical
nonhomologous end-joining (NHEJ) or the alternative
end-joining (alt-NHEJ) pathway, which require
minimal overlapping
nonhomologous chromosomes to repair double-
stranded DNA breaks (reviewed40). Using the
information derived from genomic breakpoint junction
sequence analysis and in vitro cleavage assays, the
knowledge that topoII introduces staggered nicks in
genes showed that
sequences between

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Mediterr J Hematol Infect Dis 2011; 3: Open Journal System
Figure 3. Model for formation of the t(15;17) in a case of mitoxantrone-related t-APL (case F8) following topoII induced cleavage in PML
and RARA loci with 4-base 5’ overhangs and aberrant DNA repair. Native PML and RARA sequences are red and blue, respectively. The
processing includes exonucleolytic nibbling to form two-base (der(15)) or single-base (der(17)) homologies and creation of both breakpoint
junctions by error-prone nonhomologous end-joining (NHEJ). In formation of the der(15), positions 1487-1488 on the antisense strand of
PML are lost by exonucleolytic nibbling (pink) before NHEJ joins the indicated bases. Positions 1485-1487 on the sense strand of PML are
lost by exonucleolytic nibbling (pink) and the der(17) forms by NHEJ. Template-directed polymerization of the relevant single-stranded
overhangs fills in any gaps (light blue). Each RARA overhang is preserved completely. Adapted from Mistry et al.33with permission.
DNA with 4-bp overhangs22and considering known
mechanisms of DNA repair it was possible to construct
models by which the t(15;17) may have been formed in
each case analyzed (see Figures 3 & 4). Taken
together, these data provide very strong evidence that
mitoxantrone is a causative agent in the pathogenesis
of t-APL.
Investigation of Molecular Mechanisms in t-APL
Cases Arising Following Other TopoII Poisons.
Epirubicin exposure has been linked to secondary
leukemias with a range of balanced rearrangements,
including translocations involving the MLL locus, core
binding factor leukemias and t-APL with the
t(15;17).31,41In order to gain further insights into
molecular mechanisms underlying epirubicin-related
leukemias, we characterized
breakpoint junction regions in a series of 6 t-APL cases
that arose following breast cancer therapy.42Epirubicin
was generally used as a component of combination
chemotherapy, with a median latency period from first
exposure to presentation of t-APL of 26 months (range
18-48 months). Although the number of cases
examined was small, significant breakpoint clustering
was observed in both the PML and RARA loci (P= .009
and P = .017, respectively), with PML breakpoints
t(15;17) genomic
lying outside the mitoxantrone-associated “hotspot”
region (Figure 1). Functional assays demonstrated that
recurrent breakpoints identified in the PML and RARA
loci in epirubicin-related t-APL were preferential sites
of topoII-induced DNA damage that were enhanced by
epirubicin.42Again, using the same approach as for
mitoxantrone-related t-APLs,
constructed to explain the formation of the t(15;17) in
APLs arising following epirubicin exposure.42
There also have been reports of t-APL occurring
following treatment with other topoII poisons (e.g.,
etoposide) used for lymphomas and various solid
tumors, as well as Langerhans cell histiocytosis.12,25,43
To determine whether topoII–mediated cleavage is
relevant to other drugs associated with t-APL, we also
have studied a patient in whom APL developed after
treatment for laryngeal carcinoma that included
etoposide and doxorubicin.33
metabolites and doxorubicin induced topoII to cleave
DNA at the PML and RARA translocation breakpoints.
Moreover, the cleavage sites could recombine to form
the der(15) and der(17) breakpoint junctions observed
in this patient. Taken together, these results suggest
that topoII–mediated cleavage is a general mechanism
causing DNA damage in APL that develops after
treatment with various agents that target topoII (Figure
models could be
Etoposide and its