Panhandle and reverse-panhandle PCR enable cloning
of der(11) and der(other) genomic breakpoint
junctions of MLL translocations and
identify complex translocation of
MLL, AF-4, and CDK6
Leslie J. Raffini*†, Diana J. Slater*†, Eric F. Rappaport‡, Luca Lo Nigro*§, Nai-Kong V. Cheung¶, Jaclyn A. Biegel?**,
Peter C. Nowell††, Beverly J. Lange*,**, and Carolyn A. Felix*,**‡‡
*Division of Oncology,‡Joseph Stokes, Jr. Research Institute, and?Department of Human Genetics and Molecular Biology, Children’s Hospital of
Philadelphia, Philadelphia, PA 19104; Departments of **Pediatrics and††Pathology and Laboratory Medicine, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104; and¶Department of Pediatrics, Memorial Sloan–Kettering Cancer Center, New York, NY 10021
Contributed by Peter C. Nowell, February 5, 2002
We used panhandle PCR to clone the der(11) genomic breakpoint
junction in three leukemias with t(4;11) and devised reverse-
panhandle PCR to clone the breakpoint junction of the other
derivative chromosome. This work contributes two elements to
knowledge on MLL translocations. First is reverse-panhandle PCR
for cloning breakpoint junctions of the other derivative chromo-
somes, sequences of which are germane to understanding the MLL
translocation process. The technique revealed duplicated se-
quences in one case of infant acute lymphoblastic leukemia (ALL)
and small deletions in a case of treatment-related ALL. The second
element is discovery of a three-way rearrangement of MLL, AF-4,
and CDK6 in another case of infant ALL. Cytogenetic analysis was
at relapse. Panhandle PCR analysis of the diagnostic marrow
identified a breakpoint junction of MLL intron 8 and AF-4 intron 3.
Reverse-panhandle PCR identified a breakpoint junction of CDK6
from band 7q21-q22 and MLL intron 9. CDK6 encodes a critical cell
cycle regulator and is the first gene of this type disrupted by MLL
translocation. Cdk6 is overexpressed or disrupted by translocation
in many cancers. The in-frame CDK6-MLL transcript is provocative
with respect to a potential contribution of the predicted Cdk6-MLL
fusion protein in the genesis of the ALL, which also contains an
in-frame MLL-AF4 transcript. The sequences in these three cases
show additional MLL genomic breakpoint heterogeneity. Each
breakpoint junction suggests nonhomologous end joining and is
consistent with DNA damage and repair. CDK6-MLL is a new fusion
of both genes.
in infants. The translocations fuse the breakpoint cluster region
(bcr) that spans exons 5–11 of MLL with one of many partner
genes, 31 of which have been cloned so far [J. L. Huret, (2001)
provide clues to the translocation mechanism and suggest DNA
damage and repair (4–7). Backtracking nonconstitutional MLL
translocations to the prenatal period (8–10) indicates that the
damage occurs in utero, but the agent(s) is unknown. Because
similar translocations occur in leukemias related to chemother-
apy with DNA topoisomerase II inhibitors, DNA topoisomerase
II may be implicated in the damage (reviewed in ref. 11).
Maternal prenatal consumption of dietary DNA topoisomerase
II inhibitors may increase the risk of infant acute myeloid
polymorphism is associated with infant leukemias with MLL
translocations (13), and the NQO1 substrate benzoquinone
he MLL gene was cloned 10 years ago as a common target
of translocations in human acute leukemias (1–3), especially
interferes with DNA topoisomerase II (14). A model for the
translocation process involves DNA topoisomerase II-mediated
chromosomal breakage and formation of the translocations
when the breakage is repaired.
Nonetheless, the genomic breakpoint junction sequences of
and treatment-related leukemias that represent the spectrum of
partner genes of MLL. The large number of potential partner
genes can impede genomic cloning. We have used panhandle
PCR approaches to clone the der(11) genomic breakpoint
junctions (7, 15–17). Amplification of the genomic breakpoint
junctions of the other derivative chromosomes with primers
based on der(11) sequences may be unsuccessful if there are
large duplications or deletions or if the rearrangements are
complex. Here, we developed reverse-panhandle PCR to clone
the breakpoint junctions of the other derivative chromosomes
and identified a new CDK6-MLL fusion in a cryptic, complex
IRBs at the Children’s Hospital of Philadelphia and Memorial
Sloan–Kettering Cancer Center approved this research.
Case Histories. Patient 45 was diagnosed with French–American–
British (FAB) L1 acute lymphoblastic leukemia (ALL) at age 3
weeks. She presented with hepatosplenomegaly and a WBC
count of 86 ? 109?liter, but no evidence of central nervous
system disease. The bone marrow karyotype in five metaphases
was 46,XX,t(4;11). The immunophenotype was Tdt?, CD19?,
CD10?, CD20?; no myeloid antigens were expressed. At age 5
months, a progressive seizure disorder with loss of milestones
developed. Head MRI and CT scans were normal. By age 10
months, myeloblasts in the cerebrospinal fluid suggested CNS
Abbreviations: bcr, breakpoint cluster region; ALL, acute lymphoblastic leukemia.
database (accession nos. AF487903–487906 and AF492830–AF492835).
†L.J.R. and D.J.S. contributed equally to this work.
§Present address: Division of Pediatric Hematology Oncology, University of Catania, Via S.
Sofia 78, 95123, Catania, Italy.
‡‡To whom reprint requests should be addressed at: Division of Oncology, Abramson
Research Center, Room 902B, Children’s Hospital of Philadelphia, 3516 Civic Center
Boulevard, Philadelphia, PA 19104. E-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
April 2, 2002 ?
vol. 99 ?
no. 7 www.pnas.org?cgi?doi?10.1073?pnas.062066799
relapse with lineage shift. She suffered rapid neurologic dete-
rioration and died.
Patient t-120 was diagnosed with stage IV neuroblastoma at
age 2 years. His primary posterior mediastinal tumor was
metastatic to the bone and marrow. Memorial Sloan–Kettering
N7 treatment included four cycles of cyclophosphamide, doxo-
rubicin, and vincristine, three cycles of cisplatin and etoposide
(PVP), surgical resection, local radiation, radiolabeled anti-GD2
mAb (3F8), and autologous marrow rescue with cells harvested
after chemotherapy cycle 5 (PVP) and purged ex vivo with 3F8.
Eleven months after starting treatment and 2 weeks after
transplant, the WBC count was 46 ? 109?liter and FAB L2 ALL
was diagnosed. The karyotype in 17 metaphases was
The presentation of patient 38 at infant ALL diagnosis was as
described (15). The 3-month-old girl presented with hepatospleno-
megaly and a WBC count of 399 ? 109?liter. The marrow was
replaced with FAB L1, Tdt?, CD19?, CD10?, CD20?, CD34?
blasts. Cytogenetic analysis of the diagnostic marrow was unsuc-
cessful (15). She received CCG 1883-like chemotherapy (18) but
relapsed in the marrow at age 4 years, 25 months from completion
of this treatment, when the marrow karyotype in three metaphases
was 47,XX,t(4;11)(q21;q23),del(7)(q21q31),?8. She died from
Pseudomonas sepsis during reinduction.
Detection of MLL Gene Rearrangements. Rearrangements were
examined by Southern blot analysis of BamHI-digested DNA
with the B859 fragment of ALL-1 cDNA (1).
Cloning of der(11) Genomic Breakpoint Junctions. For the leukemia
breakpoint junction was described (GenBank accession no.
AF031403) (15). The der(11) genomic breakpoint junctions in
the leukemias of patients 45 and t-120 were amplified by
panhandle PCR as described (15), except that primers 3 and 4
were those used for cDNA panhandle PCR (7). Panhandle PCR
products were subcloned by recombination PCR (7); subclones
were screened by PCR and sequenced. der(11) breakpoint
junctions were confirmed by amplification of genomic DNAs
with MLL- and AF-4-specific primers and direct sequencing.
Cloning of Genomic Breakpoint Junctions of Other Derivative
Chromosomes. Reverse-panhandle PCR was accomplished by
ligation of a phosphorylated oligonucleotide containing known
sense sequence from MLL intron 10?exon 11 to the 3? ends of
BamHI-digested DNA and formation of a stem-loop template
from the antisense strand. The template contained unknown
partner sequence, the breakpoint junction of the other derivative
its complement at either end of the ‘‘handle’’ enabled amplifi-
cation of the breakpoint junction in three sequential, single-
primer, two-sided PCRs with primers all antisense with respect
to MLL exon 11 or intron 10?exon 11 sequences (Fig. 1).
In Step 1, 2.5 ?g of genomic DNA was digested with 20 units
of BamHI (New England Biolabs). The DNA was treated with
0.025 units of calf intestinal alkaline phosphatase (Boehringer
Mannheim) at 37°C for 30 min and purified with a Geneclean III
kit (Bio 101). In Step 2, a single-stranded, 5? phosphorylated
GTTCGCTGTAAGAGC-3?) was ligated to the 3? ends. The
4-base 5? end of the oligonucleotide was complementary to the
5? overhang of the BamHI-digested DNA; its 3? end corre-
11 (GenBank accession no. U04737). Each 50-?l ligation reac-
tion mixture contained 2.5 ?g of DNA, a 50-fold molar excess of
5? phosphorylated oligonucleotide, 1 Weiss unit of T4 DNA
ligase, and 1? ligase buffer (Boehringer Mannheim). Ligations
were performed at 4°C. The DNA was purified with a Geneclean
III kit (Bio 101). The stem-loop template was formed from the
antisense strand in Step 3. After heating the other components
to 80°C for 5 min, 20 ng of digested, ligated DNA was added to
1.75 units of Taq?Pwo DNA polymerase mix, 368 ?M each
dNTP, and 1.05? PCR buffer (Expand Long Template PCR
System; Boehringer Mannheim) in a 47.5-?l reaction mixture.
The DNA was made single-stranded by heating the reaction
mixture at 94° for 1 min. The template was generated by a 2-min
ramp to 72°C and incubation at 72°C for 30 s to promote
intrastrand annealing of the ligated oligonucleotide to the com-
plementary sequence in the antisense strand and polymerase
extension of the recessed 3? end (15). In Step 4, primer 1
extension made the template double-stranded, allowing expo-
nential amplification with primer 1, which anneals to both ends.
After 2.5 ?l of a 5-pmol??l solution of primer 1 corresponding
to MLL exon 11 antisense positions 8342–8315 (5?-GGATCCA-
CAGCTCTTACAGCGAACACAC-3?) was added, each final,
50-?l PCR contained 12.5 pmol of primer 1, 350 ?M each dNTP,
and 1? PCR buffer. After denaturation at 94°C for 1 min, 10
cycles at 94°C for 10 s and 68°C for 7 min and 20 cycles at 94°C
for 10 s and 68°C for 7 min (increment, 20 s?cycle) were used,
followed by final elongation at 68°C for 7 min. Steps 5 and 6 were
sequential, two-sided, single-primer-nested PCRs with primers 2
11 positions 8336–8305 (5?-ACAGCTCTTACAGCGAACA-
CACTTGGTACAGA-3?) and MLL exon 11?intron 10 positions
GATCTAGA-3?). Nested PCR conditions were the same as in
the initial PCR; 1-?l aliquots of the products of respective
preceding PCRs were used as templates.
Reverse-panhandle PCR products were subcloned by recom-
bination PCR (7). pUC19 was linearized by HindIII digestion.
MLL ends complementary to the ends of the products of
the last nested PCR in reverse-panhandle PCR were added
to the linearized vector by amplification with primers 5?-
other derivative chromosomes of MLL translocation.
Reverse-panhandle PCR analysis of genomic breakpoint junction of
Raffini et al.
April 2, 2002 ?
vol. 99 ?
no. 7 ?
ATGGTCATAGC-3? and 5?-ACCAAGTGTGTTCGCTG-
The breakpoint junctions of the other derivative chromosomes
were validated by PCR amplification of genomic DNA with
gene-specific primers and direct sequencing.
PCR was performed on genomic DNA from the diagnostic
marrow of patient 38 with the sense primer 5?-GAAATGGGT-
GCAGTGTTCCA-3? from AF-4 intron 3 and the antisense
primer 5?-TGGATTACGGGATAGGGACA-3? from CDK6 in-
tron 2 to determine whether a reciprocal AF-4-CDK6 rearrange-
ment had occurred.
Analysis of Fusion Transcripts. Reverse transcriptase–PCR analysis
of the MLL-AF-4 transcript in the marrow cells of patient 38 at
diagnosis has been described (GenBank accession no.
AF031404) (15). The same method was used to generate ran-
domly primed, first-strand cDNA and characterize the der(11)
transcript in the ALL cells of patient 45 (15). The der(11)
transcript in the ALL cells of patient t-120 was identified by
same first-strand cDNA with MLL- and AF-4-specific primers.
der(4) transcripts in the leukemia cells of patients 45 and t-120
were identified by amplification of the above-generated first-
strand cDNAs with the AF-4 exon 3 sense primer 5?-
CTCCCCTCAAAAAGTGTTGC-3? (GenBank accession no.
L13773) and the MLL exon 9 antisense primer 5?-CAATTT-
TCCAGCTGGTCCTC-3? (GenBank accession no. L04284).
The CDK6-MLL transcript was identified in the ALL cells of
patient 38 with the sense primer 5?-CGTGGTCAGGTTGTT-
TGATG-3? from CDK6 exons 1–2 (GenBank accession no.
NM?001259) and the MLL exon 13 antisense primer 5?-
GCCGCTCAGTACAGTTCACA-3?. PCR was performed with
the same first-strand cDNA and the AF-4 exon 3 sense primer
5?-CTCCCCTCAAAAAGTGTTGC-3? and the CDK6 exon 4
antisense primer 5?-GACTTCGGGTGCTCTGTACC-3?.
Characterization of der(11) and der(4) Genomic Breakpoint Junctions
and Fusion Transcripts in Infant ALL Cells. Southern blot analysis
revealed 6.8- and 2.1-kb MLL bcr rearrangements in the infant
ALL of patient 45 (Fig. 2A). Panhandle PCR amplified the
der(11) genomic breakpoint junction (Fig. 2 A and B). The
6,808-bp product suggested that the 6.8- and 2.1-kb rearrange-
ments were from the der(11) and der(4) chromosomes, respec-
tively. The MLL der(11) breakpoint was 3? in the bcr at position
6775 in intron 8 (GenBank accession no. U04737) (Fig. 2B). The
der(11) breakpoint in the partner gene was position 34744 in
AF-4 intron 3 (GenBank accession no. AJ238093).
AF-4 and MLL primers were designed to amplify the der(4)
genomic breakpoint junction predicted by the der(11) sequence.
The expected product size was 494 bp but a ?1.2-kb product was
obtained (data not shown). Reverse-panhandle PCR was tested
in this case with a known der(4) breakpoint junction sequence.
The 2,232-bp product was consistent with the 2.1-kb MLL bcr
the same as in the PCR product obtained with gene-specific
primers, validating reverse-panhandle PCR as a cloning strategy
for the breakpoint junction of the other derivative chromosome
of an MLL translocation. The AF-4 der(4) breakpoint was
position 34864 or 34865 in intron 3; the MLL der(4) breakpoint
was position 6166 or 6167 in intron 8. ‘‘A’’ residues at the
breakpoints in both genes precluded more precise breakpoint
assignments (Fig. 2C). Depending on the exact breakpoint
positions, 609–610 bases from AF-4 and 121–122 bases from
MLL were present in both derivative chromosomes, suggesting
duplication (Fig. 2 B and C). Other identical 1- to 4-base
sequences in MLL and AF-4 were present near the der(11) and
der(4) breakpoint junctions. There were AluJo repeats in MLL
and AF-4, within ?1,681 bp and ?259 bp, respectively, of the
der(11) breakpoint junction (Fig. 2B). The MLL der(4) break-
point was within an AluY, and there was an AluY in AF-4 intron
3 ?988 bp from the der(4) breakpoint junction (Fig. 2C).
der(11) and der(4) transcripts were produced. The der(11)
transcript fused MLL exon 8 in-frame to AF-4 exon 4. The der(4)
transcript fused AF-4 exon 3 in-frame to MLL exon 9.
Characterization of der(11) and der(4) Genomic Breakpoint Junctions
and Fusion Transcripts in Treatment-Related ALL Cells. Southern blot
analysis revealed 7.2- and 2.0-kb MLL bcr rearrangements in the
treatment-related ALL of patient t-120 (Fig. 3A). Panhandle
PCR products (7,295 bp) suggested that the 7.2- and 2.0-kb
rearrangements were from the der(11) and der(4) chromosomes,
respectively (Fig. 3A). The MLL der(11) breakpoint was position
6588 or 6589 in intron 8, also 3? in the bcr; the der(11) breakpoint
in the partner gene was AF-4 intron 3 position 7130 or 7131 (Fig.
3B). These breakpoints could not be localized precisely because
both genes contain an ‘‘A’’ residue at the breakpoint junction.
panhandle PCR (Left) and reverse-panhandle PCR (Right) products. The
8.3-kb fragment on Southern blot is from unrearranged MLL allele (Center,
dash); arrows show rearrangements. (B) Summary of der(11) genomic
breakpoint junction in recombination PCR-generated subclones from pan-
handle PCR. One subclone was sequenced in its entirety; the breakpoint
junction was verified in three more subclones. The 5? 6,639 bp include MLL
primer 4 and MLL bcr sequence. Ninety-six base pairs of 3? sequence are
AF-4 DNA. Seventy-three base pairs of 3? sequence extend from ligated
oligonucleotide (P-Oligo) through MLL primer 3 (Top). Arrow shows MLL
and AF-4 breakpoint positions (Bottom). Underlines show short homolo-
gies (bottom). Repetitive sequences are shown (Middle). (C) Summary of
der(4) genomic breakpoint junction in recombination PCR-generated sub-
clone from reverse-panhandle PCR. In reverse-panhandle PCR, nested
primer 3 from MLL exon 11?intron 10 anneals to both ends of the template.
Thirty-five base pairs of 5? sequence extend from MLL primer 3 through
ligated oligonucleotide (P-Oligo). Twenty-nine to 30 bp of 5? sequence are
AF-4. The 3? 2167–2168 bp are MLL bcr sequence through MLL primer 3
(Top). Arrowheads show AF-4 and MLL breakpoint positions; ‘‘A’’ residue
in both genes precluded precise assignments (Bottom). Short homologies
are underlined (Bottom). Repetitive sequences are shown (Middle). One
subclone was sequenced in its entirety; three PCRs with gene-specific
primers confirmed der(4) breakpoint junction.
(A) MLL bcr rearrangements in ALL of patient 45 (Center) and
www.pnas.org?cgi?doi?10.1073?pnas.062066799Raffini et al.
Other 1- to 4-base homologies were present near the breakpoints
in both genes (Fig. 3B). The MLL and AF-4 der(11) breakpoints
were near AluJo and other repetitive sequence elements.
The 2,079-bp reverse-panhandle PCR product was consistent
with the 2.0-kb rearrangement on the Southern blot (Fig. 3A).
The AF-4 der(4) breakpoint was position 7108, 7109, or 7110 in
intron 3; the MLL der(4) breakpoint was position 6594, 6595, or
6596 in intron 8 (Fig. 3C). In addition to the 5?-CA-3? immedi-
ately at the breakpoints in both genes that precluded more
precise assignments, other short, homologous sequences flanked
the der(4) breakpoint junction (Fig. 3C). The closest repetitive
sequences to the der(4) breakpoints in both genes were MERs
(Fig. 3C). Depending on the exact breakpoint positions, 4–7 bp
from MLL and 19–22 bp from AF-4 were lost during the
cDNA panhandle PCR identified an in-frame der(11) tran-
script joining MLL exon 8 to AF-4 exon 4. A der(4) transcript
fusing AF-4 exon 3 in-frame to MLL exon 9 also was produced.
Reverse-Panhandle PCR Identifies Complex Translocation of MLL,
AF-4, and CDK6. Southern blot analysis of the diagnostic marrow
of patient 38 revealed 7.0- and 2.0-kb MLL bcr rearrangements
(Fig. 4A) (15). Although cytogenetic analysis was unsuccessful,
panhandle PCR identified an MLL intron 8-AF-4 intron 3
genomic breakpoint junction of a putative der(11) chromosome.
The MLL breakpoint was position 3802 in intron 8; the AF-4
breakpoint was position 16039 in intron 3 (GenBank accession
no. AF031403) (15). Because PCR with AF-4- and MLL-specific
primers designed from this sequence did not identify the pre-
dicted der(4) breakpoint junction (data not shown), reverse-
panhandle PCR was used to identify the genomic breakpoint
junction of the other derivative chromosome, the presence of
which was suggested by the two MLL bcr rearrangements on the
Southern blot. The panhandle PCR product size suggested that
the 7.0-kb rearrangement was from the putative der(11) chro-
mosome (15). The Southern blot and panhandle PCR product
size predicted a reverse-panhandle PCR product of ?2.0 kb. A
2,241-bp reverse-panhandle PCR product was obtained (Fig.
4A); the sequence showed that the 3? portion of the MLL bcr had
not fused with AF-4 but with the CDK6 (cyclin-dependent kinase
6) gene from chromosome band 7q21-q22 (Fig. 4B). The CDK6
genomic clone AC004128. The MLL breakpoint was position
7156–7158 in intron 9. A total of 3355–3357 were lost from MLL
in CDK6 and MLL precluded more precise breakpoint assign-
ments. Other short, homologous sequences in CDK6 and MLL
flanked the breakpoint junction (Fig. 4B). An MLL exon 8-AF-4
exon 4 fusion transcript was produced as described previously
(GenBank accession no. AF031404) (15). The CDK6-MLL re-
arrangement produced an in-frame fusion transcript of CDK6
exon 2 with MLL exon 10 (Fig. 4C). Although there were no
mitoses on cytogenetic analysis of the diagnostic bone marrow
(15), the bone marrow karyotype at relapse demonstrated
del(7)(q21q31) in addition to the t(4;11) (Fig. 5). No material
was available for fluorescence in situ hybridization analysis, but
the molecular analyses of the diagnostic marrow are consistent
with a three-way translocation. Genomic DNA from the diag-
nostic marrow was analyzed with AF-4- and CDK6-specific
primers, but no reciprocal AF-4-CDK6 product was obtained. In
addition, no AF-4-CDK6 fusion transcript was detected (data not
Examination of the genomic breakpoint junctions of both de-
rivative chromosomes is essential to understanding the MLL
translocation process. It is customary to attempt isolation of the
genomic breakpoint junction of the other derivative chromo-
some by PCR with partner gene- and MLL-derived primers
designed based on the der(11) sequence (5, 7) or, in the case of
the t(4;11), based on karyotypic evidence of potential involve-
ment of a known partner gene of MLL (4). This approach fails
when there are large duplications, deletions, inversions, or
complex rearrangements or when the der(11) sequence or the
partner gene is unknown. Because MLL has many partner genes,
we previously implemented panhandle PCR and panhandle
variant PCR for der(11) genomic breakpoint junctions and
cDNA panhandle PCR for der(11) transcripts in which all
we devised a reverse-panhandle PCR approach to clone the
genomic breakpoint junctions of the other derivative chromo-
variant PCR (7, 9, 15, 17). Stem-loop templates are created in all
three genomic methods by BamHI digestion, which creates a
fragment size amenable to PCR, and ligation of known MLL bcr
sequence to the unknown partner sequence in the fragment (7,
fragment on Southern blot (Center, dash) and larger, 8.3-kb panhandle PCR
product (Left) are from the unrearranged MLL allele; arrows show rearrange-
ments (Center). (B) Summary of der(11) genomic breakpoint junction in
recombination PCR-generated subclones from panhandle PCR. One subclone
was sequenced in its entirety; the breakpoint junction was verified in another
subclone. The 5? 6431–6432 bp include MLL primer 4 and MLL bcr sequence.
790–791 bp of 3? sequence are AF-4. Seventy-three base pairs of 3? sequence
extend from ligated oligonucleotide (P-Oligo) through MLL primer 3 (Top).
Arrowheads show AF-4 and MLL breakpoint positions; ‘‘A’’ residue in both
genes precluded precise assignments (Bottom). Underlines indicate short
homologies (Bottom). Repetitive sequences are shown (Middle). (C) Summary
of der(4) genomic breakpoint junction in recombination PCR-generated sub-
clones from reverse-panhandle PCR. One subclone was sequenced in its en-
tirety; the breakpoint junction was verified in three more subclones. Thirty-
five base pairs of 5? sequence extend from MLL primer 3 through ligated
oligonucleotide (P-Oligo). 304–306 bp of 5? sequence are AF-4. The 3? 1738–
1740 bp include MLL bcr sequence through MLL primer 3. Arrowheads show
AF-4 and MLL breakpoint positions; ‘‘CA’’ in both genes precluded precise
assignments (Bottom). Short homologies are underlined (Bottom). Repetitive
sequences are shown (Middle).
(A) MLL bcr rearrangements in ALL of patient t-120 (Center) and
Raffini et al.
April 2, 2002 ?
vol. 99 ?
no. 7 ?
9, 15–17). All four panhandle PCR approaches lead readily to
known and unknown partner sequences.
Although both the der(11) and der(4) genomic breakpoint
junctions have been amplified with gene-specific primers in
many de novo leukemias with t(4;11) (4, 5), both genomic
breakpoint junctions have been characterized in few de novo
leukemias with other MLL translocations (6) and few leukemias
after chemotherapy with DNA topoisomerase II inhibitors (7,
21–23). As in the ALL of patient 45, the sequences in de novo
cases have shown regions up to several hundred bases from MLL
and?or the partner gene on both derivative chromosomes,
suggesting duplication (4–6). Deletions of several hundred bases
also have been observed (4, 6). Except in the ML-1 cell line (22),
in treatment-related leukemias with MLL translocations includ-
ing the ALL of patient t-120, the sequences suggest more precise
recombinations with deletions or duplications of relatively few
bases (7, 21, 23).
The association of DNA topoisomerase II-targeted chemo-
therapy with leukemias with MLL translocations has suggested
that repair of DNA topoisomerase II-mediated chromosomal
breakage may cause the translocations (reviewed in ref. 11).
Sequence information on both genomic breakpoint junctions
may suggest different types of DNA topoisomerase II cleavage
in de novo and treatment-related cases. DNA topoisomerase II
creates 4-base, staggered, double-stranded breaks in DNA, but
DNA topoisomerase II also introduces single-stranded nicks as
kinetic intermediates of double-stranded breaks (24). The more
precise recombination in treatment-related cases may be more
consistent with the processing of 4-base, staggered, double-
stranded breaks (23). Duplicated sequences in the de novo cases
may arise from staggered, single-stranded nicks in both DNA
strands and template-directed polymerization of the intervening
sequence (4, 5). The large, deleted regions in some de novo cases
may arise from multiple breaks or extensive processing (4, 6).
Sequence differences between treatment-related and de novo
cases may suggest that different types of breakage are induced
in the leukemias studied here, as those at other MLL genomic
breakpoint junctions (4–7, 9, 15), may suggest that nonhomolo-
gous end-joining is involved in the repair (4, 23).
The MLL-AF-4 genomic breakpoint junction in the ALL of
patient 38 predicted a reciprocal AF-4-MLL genomic breakpoint
junction, but reverse-panhandle PCR led instead to the discov-
ery of the CDK6-MLL junction in a cryptic, complex, three-way
rearrangement. The ?226-kb CDK6 gene located at chromo-
some 7q21-q22 contains 7 exons (25), which encode a 325-aa,
40-kDa protein (26, 27). Cdk6 is a D cyclin-dependent kinase
signaling at the G1?S cell cycle transition and the major Cdk in
human lymphoid cells (27–29). Upon D cyclin activation, Cdk6
phosphorylates and inactivates Rb, inhibiting its growth-
suppressive function, activating E2F transcription factors, and
enabling entry into S phase (30). An in-frame CDK6-MLL fusion
transcript was identified. The corresponding full-length fusion
The 8.3-kb fragment (Left) was from unrearranged MLL allele; 7.0-kb fragment was from MLL-AF-4 rearrangement (15). (B) Sequence of genomic breakpoint
junction of other derivative chromosome in recombination PCR-generated subclone from reverse-panhandle PCR. Thirty-five base pairs of 5? sequence are from
MLL primer 3 through ligated oligonucleotide (P-Oligo). 1028–1030 bp of 5? sequence are CDK6. The 3? 1176–1178 bp include MLL bcr sequence from intron 9
through MLL primer 3. Arrowheads show CDK6 and MLL breakpoint positions; ‘‘AG’’ in both genes precluded precise assignments (Bottom). Short homologies
are underlined (Bottom). Repetitive sequences are shown (Middle). (C) Detection of CDK6-MLL fusion transcript. Reverse transcriptase–PCRs with CDK6 exons
1–2 and MLL exon 13 primers and randomly primed first-strand cDNA gave a 548-bp product (Top). Reaction with ?-actin primers and RNA-negative reagent
control reaction (dH2O) were performed (Top). Sequencing showed in-frame fusion of CDK6 exon 2 at position 486 of the 1,249-bp CDK6 cDNA (GenBank
accession no. NM?001259) to MLL exon 10 (Bottom). (D) Cdk6 and MLL proteins and predicted Cdk6-MLL fusion protein.
(A) MLL bcr rearrangements in ALL of patient 38 (15) (arrows, Left) and reverse-panhandle PCR product (Right) consistent with 2.0-kb rearrangement.
The karyotype was described as 47,XX,t(4;11)(q21;q23),del(7)(q21q31),?8.
www.pnas.org?cgi?doi?10.1073?pnas.062066799Raffini et al.
transcript would include the first 123 codons of CDK6 and the
last 2,476 codons of MLL. The PLSTIRE helix ?1, ? sheets,
catalytic cleft, and 22 residues from the carboxyl-terminal
?-helices of Cdk6 (26) and the zinc fingers, transactivation, and
protein (Fig. 4D). Although the term ‘‘partner gene’’ generally
refers to the gene whose 3? sequence is fused to the 5? sequence
of MLL, CDK6 is the first gene of this type identified in an MLL
MLL translocations are thought to be leukemogenic by pro-
ducing chimeric oncoproteins from the der(11) chromosome in
which the amino terminus of MLL is joined to the carboxyl
terminus of the partner protein (31). Murine models have used
5?-MLL-partner-3? constructs to establish that MLL transloca-
tions are leukemogenic (31). Latency to leukemia in these
models suggests that additional alterations also may be impor-
tant. At least one such construct (5?-MLL-FBP17-3?) shows
minimal transformation in serial replating assays (32). However,
experiments on the potential functional contribution of partner-
MLL fusion proteins have not been performed. Although an
MLL-AF-4 transcript was produced in the leukemia cells of
patient 38 (15) and the der(11) gene product is considered
critical in leukemogenesis (31), it is possible that the Cdk6-MLL
fusion protein predicted by the maintenance of a productive
ORF in the fusion transcript may have contributed as well.
Cdk6 overexpression occurs in T cell lymphoblastic lym-
phoma, T cell ALL, natural killer?T cell nasal lymphoma, and
glioblastoma multiforme (27, 33, 34). B cell splenic lymphomas
with villous lymphocytes are characterized by t(2;7)(p12;q21)
juxtaposing CDK6 to the Ig ? gene (35). A t(7;21) disrupting
CDK6 was observed in a splenic marginal zone lymphoma (35).
The central role of Cdk6 in cell cycle progression and its
recurrent alteration in human cancer suggest that the CDK6-
MLL juxtaposition may have been a cooperating mutation in
leukemogenesis in patient 38. G-banded and spectral karyotype
analyses identified t(7;11)(q22;q23) in infant ALL, MDS, and
non-Hodgkin lymphoma [F. Mitelman, B. Johansson, and F.
Mertens (2000) http:??cgap.nci.nih.gov?Chromosomes?
Mitelman], possibly suggesting that CDK6-MLL junctions will be
found in other cases.
Genomic breakpoint junction sequences and the fusion tran-
scripts resulting from three-way rearrangements provide insights
into the translocation process and molecular alterations leading to
leukemias with MLL translocations. Other three-way MLL trans-
locations have been identified (32, 36–39). Reverse-panhandle
other derivative chromosomes and is well suited to complex,
patient 45, small deletions in the treatment-related ALL of patient
t-120, and complex translocation in the ALL of patient 38 indicate
heterogeneity in MLL genomic breakpoint junctions and are con-
sistent with DNA damage and repair.
We thank Nancy Spinner for cytogenetic analysis of relapse marrow of
patient 38. C.A.F. was supported by National Institutes of Health Grants
CA66140, CA80175, CA77683, and CA85469, a Leukemia and Lym-
phoma Society Translational Research Award, and the Joshua Kahan
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