The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia.

Abstract

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

Full text

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doi:10.1182/blood-2010-11-318873
Prepublished online August 30, 2011;
2011 118: 4169-4173




James R. Downing, Charles G. Mullighan and A. Thomas Look
Silverman, Stuart S. Winter, Stephen P. Hunger, Stephen E. Sallan, Shan Zha, Frederick W. Alt,
Zhang, Alexei Protopopov, Lynda Chin, Suzanne E. Dahlberg, Donna S. Neuberg, Lewis B.
Alejandro Gutierrez, Alex Kentsis, Takaomi Sanda, Linda Holmfeldt, Shann-Ching Chen, Jianhua
subtypes of T-cell acute lymphoblastic leukemia
tumor suppressor is mutated across the major molecular
BCL11B
The

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LYMPHOID NEOPLASIA
Briefreport
TheBCL11Btumorsuppressorismutatedacrossthemajormolecularsubtypesof
T-cellacutelymphoblasticleukemia
Alejandro Gutierrez,1,2Alex Kentsis,1,2Takaomi Sanda,1Linda Holmfeldt,3Shann-Ching Chen,3Jianhua Zhang,4
Alexei Protopopov,4Lynda Chin,4,5Suzanne E. Dahlberg,6Donna S. Neuberg,6Lewis B. Silverman,1,2Stuart S. Winter,7
Stephen P. Hunger,8Stephen E. Sallan,1,2Shan Zha,9Frederick W.Alt,10James R. Downing,3Charles G. Mullighan,3and
A. Thomas Look1,2
1Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA;2Division of Hematology/Oncology, Children’s Hospital, Boston, MA;
3Department of Pathology, St Jude Children’s Research Hospital, Memphis, TN;4Belfer Institute forApplied Cancer Science, and Departments of5Medical
Oncology and6Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA;7Department of Pediatrics, University of New Mexico Health
Sciences Center,Albuquerque, NM;8Section of Pediatric Hematology/Oncology, University of Colorado School of Medicine and The Children’s Hospital,Aurora,
CO;9Departments of Pathology and Pediatrics, Institute for Cancer Genetics, Columbia University Medical Center, New York, NY; and10Howard Hughes
Medical Institute, Children’s Hospital, Immune Disease Institute and Harvard Medical School, Boston, MA
The BCL11B transcription factor is required
for normal T-cell development, and has re-
cently been implicated in the pathogenesis
of T-cell acute lymphoblastic leukemia
(T-ALL) induced by TLX overexpression or
Atmdeficiency.Tocomprehensivelyassess
the contribution of BCL11B inactivation to
humanT-ALL,weperformedDNAcopynum-
berandsequencinganalysesofT-ALLdiag-
nostic specimens, revealing monoallelic
BCL11Bdeletionsormissensemutationsin
9% (n ? 10 of 117) of cases. Structural ho-
mology modeling revealed that several of
the BCL11B mutations disrupted the struc-
ture of zinc finger domains required for this
transcription factor to bind DNA. BCL11B
haploinsufficiency occurred across each of
the major molecular subtypes of T-ALL, in-
cluding early T-cell precursor, HOXA-
positive,LEF1-inactivated,and TAL1-
positivesubtypes,whichhavedifferentiation
arrest at diverse stages of thymocyte devel-
opment. Our findings provide compelling
evidence that BCL11B is a haploinsufficient
tumor suppressor that collaborates with all
major T-ALL oncogenic lesions in human
thymocyte transformation. (Blood. 2011;
118(15):4169-4173)
Introduction
T-cell acute lymphoblastic leukemia (T-ALL) can be subclassified
into distinct molecular subtypes based on dominant oncogenic
alterations that lead to differentiation arrest at specific stages of
T-cell development.1,2These include the HOXA/MEIS1, TLX1/3,
and TAL1-overexpressing subtypes,2,3the LEF1-inactivated sub-
type,4and the recently identified group of T-ALL cases with
developmental arrest at very early stages of T-cell development
defined by a characteristic early T-cell precursor (ETP) phenotype
or by absence of biallelic TCR? deletions (ABD).5,6On the other
hand, other T-ALL oncogenic alterations, such as deletions of
CDKN2A or mutations of NOTCH1 and PTEN, occur across all
T-ALL subtypes,2,7suggesting that they can contribute to thymo-
cyte transformation at diverse stages of T-cell development.
The BCL11B transcription factor plays key roles in normal
T-cell development. In murine thymocytes, Bcl11b inactivation
leads to developmental arrest at a DN2-DN3 stage,8-10acquisition
of NK-like features,8,11and aberrant self-renewal activity.10In
humanT-ALL,BCL11Bisinvolvedinrecurrentcryptict(5;14)(q35;
q32) translocations with the TLX3 locus, in which BCL11B gene
regulatory elements drive aberrant overexpression of the TLX3
oncogene.12-15However, several lines of evidence suggest that
BCL11B haploinsufficiency may also be an important pathogenetic
consequence of this translocation. For example, we have recently
identified monoallelic Bcl11b deletions in most T-ALLs arising in
Atm-deficient mice,16and Bcl11b has been shown to suppress
murineT-lymphoblastic malignancies induced by p53 haploinsuffi-
ciency, radiation, or the BCR-ABL oncogene.17,18Furthermore,
recent work has also revealed monoallelic BCL11B lesions in
TLX1-induced murine T-ALL, and in TLX1- or TLX3-overexpressing
humanT-ALL.19
Here, we show results of array CGH and sequencing analyses in
T-ALL, which reveal heterozygous BCL11B mutations and dele-
tions across each of the major molecular subtypes of T-ALL,
indicating that BCL11B is a haploinsufficient tumor suppressor that
can collaborate with diverse oncogenic lesions during human
thymocyte transformation.
Methods
Patient samples
T-ALL diagnostic specimens were collected with informed consent in
accordance with the Declaration of Helsinki and IRB approval from a
cohort of children treated on Children’s Oncology Group (COG) P9404 and
Dana-Farber Cancer Institute (DFCI) 00-01 clinical trials (n ? 47),4,6,7as
well as from a second cohort of independent samples from St Jude
Submitted November 15, 2010; accepted July 27, 2011. Prepublished online as
BloodFirstEditionpaper,August30,2011;DOI10.1182/blood-2010-11-318873.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
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Children’s Research Hospital (SJCRH), COG AALL0434, and Associazi-
one Italiana Ematologia Oncologia Pediatrica (AIEOP) clinical trials
(n ? 70; J.R.D. and C.G.M., manuscript submitted, May 2011).
DNA copy number and expression analysis
DNA copy number was assessed by microarray-based CGH in the first
cohort of cases,4,6,7and by whole-genome sequencing or SNP array in the
second cohort (J.R.D. and C.G.M., manuscript submitted, May 2011). Gene
expression was assessed using Affymetrix U133 Plus 2.0 microarrays.
Complete DNA copy number and expression analysis is available in
supplementalMethods(availableontheBloodWebsite;seetheSupplemen-
tal Materials link at the top of the online article) and are available in the
NCBI GEO website under accession number GSE14618 and GSE28703.
Mutation detection
Sequencing of the entire coding region of BCL11B together with key exons
of NOTCH1, FBXW7, PTEN, PIK3CA, PIK3RA, AKT1-3, NRAS, KRAS,
and LEF1, was performed by PCR amplification and Sanger sequencing, as
previously described.4,7
Structural homology modeling
Structural homology modeling was used to model BCL11B tandem
ZF2-ZF3 zinc finger binding to a canonical GC-rich DNA oligonucleotide
sequence, based on the high-resolution crystal structure of Egr1 (Zif268) in
complex with DNA.20This structural model was calculated using SWISS-
MODEL,21producing a high quality structure with a model-template C-?
root mean square deviation of 2.9Angstroms.
Statistical analysis
Differences in continuous data were assessed via the Mann-Whitney
(rank-sum) test. Differences in event-free and overall survival were
assessed by the log-rank test, and time-to-event distributions were plotted
via the Kaplan-Meier method.
Results and discussion
To further define the role of BCL11B inactivation in human T-ALL,
we analyzed DNAcopy number at the BCL11B locus in a cohort of
primary T-ALL lymphoblast specimens, identifying monoallelic
BCL11B deletions in 6% of cases (n ? 3 of 47). These included
1 microdeletion within the BCL11B locus, 1 small deletion
involving BCL11B and 6 additional genes, and 1 large 26 Mbp
deletionofthedistalarmofchromosome14(Figure1A,supplemen-
tal Figure 1). BCL11B resequencing was performed in 43 of these
cases together with an additional cohort of 70 T-ALL specimens
with matched germ line DNA, revealing heterozygous missense
BCL11B mutations in an additional 7 cases, as well as in 19%
(n ? 3 of 16) of T-ALL cell lines (Figure 1B). None of these
represent known single nucleotide polymorphisms based on the
NCBI (dbSNP131) or the 1000 Genomes databases (accessed
November 12, 2010), and we confirmed that BCL11B mutations
were somatically acquired in the 3 cases in which germ line DNA
was available. Taken together, we thus identified monoallelic
BCL11B lesions in 9% (n ? 10 of 117) of primary T-ALL patient
samples.
BCL11B is a zinc finger transcription factor that binds DNAvia
its Cys2His2zinc finger domains.22,23Eight of the 11 BCL11B
missense mutations we identified, including 6 of the 7 in primary
patient samples, affected residues within BCL11B zinc finger
domains. To determine whether these mutations might disrupt zinc
finger domain-mediated transcriptional activity, structural homology
modeling of canonical DNA binding of the BCL11B zinc fingers
was performed based on the high-resolution crystal structure of the
zinc finger domains of the Egr1 (Zif268) transcription factor in
complex with DNA.20Strikingly, all but 1 of the mutations
identified within ZF2-ZF3 zinc finger domains disrupted conserved
amino acids modeled to be required for the structural stability of
the zinc finger domain or its binding to DNA (Figure 1E-G and
supplemental Figure 2; mutation E532K was of indeterminate
predicted phenotype).
To identify characteristics associated with BCL11B inactivation,
we analyzed gene expression data we had available on 110 of the
117 patient samples analyzed in our study, including 8 of the
9 cases with BCL11B genetic alterations, which revealed BCL11B
haploinsufficiency across the major molecular subtypes of T-ALL,
including the early T-cell precursor (ETP), HOXA/MEIS1-positive,
LEF1-inactivated, and TAL1-positive cases (Figure 2A-B). These
molecular subtypes of T-ALL are associated with differentiation
arrest at distinct stages of T-cell development,3-6thus suggesting
that the pathogenic role of BCL11B haploinsufficiency is not solely
because of induction ofT-cell differentiation arrest, which occurs at
the DN2-DN3 stage on complete BCL11B inactivation in normal
thymocytes.8,10
To identify candidate therapeutic targets in BCL11B-mutant
T-ALL, we also performed gene set enrichment analysis (GSEA),
which revealed an inverse association between BCL11B-mutant
T-ALL and genes down-regulated by rapamycin treatment in the
BJAB Burkitt lymphoma cell line (supplemental Figure 3),24
suggesting the need for additional studies to evaluate the potential
therapeutic utility of mTOR inhibitors in BCL11B-mutant T-ALL.
Low BCL11B expression predicted failure of induction chemo-
therapy, but we were unable to identify a BCL11B expression
threshold that accurately predicted long-term outcome, while
BCL11B mutations/deletions similarly did not predict treatment
response (supplemental Figure 4).
Recurrent cryptic t(5;14)(q35;q32) translocations juxtaposing
BCL11B and TLX3 have been described in T-ALL, which result in
BCL11B gene regulatory elements driving the aberrant over-
expression of TLX3.12-15,25These translocations have been thought
to be pathogenic because of the resultant overexpression of the
TLX3 oncogene. However, our data suggest that the inactivation of
BCL11B is also an important pathogenic consequence of these
translocations. Our findings provide compelling evidence that
BCL11B is a haploinsufficient tumor suppressor across the major
molecular subtypes of T-ALL, which are blocked at diverse stages
of thymocyte development, suggesting that BCL11B loss may have
pathogenic roles extending beyond differentiation arrest.
Acknowledgments
The authors would like to gratefully acknowledge the children with
T-ALL and their families, as well as members of the COG, DFCI,
SJCRH, andAIEOP member institutions, for the samples analyzed
in these studies.
This work was funded by National Institute of Health grants
2P01CA109901 (A.T.L. and F.W.A.), 5P01CA68484 (S.E.S. and
A.T.L.), and 1RO1CA158073 (S.Z.). COG cell banking and sample
distribution were supported by the COG 9900 cell biology study
and grants CA98543, CA114766, CA98413, CA30969, and
CA29139 from the National Institutes of Health. Sequence and
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analysis of St Jude, AEIOP, and COG samples was supported by
the St Jude Children’s Research Hospital–Washington University
Pediatric Cancer Genome Project. A.G. is supported by National
Institutes of Health grant 1K08CA133103 and is a scholar of the
AmericanSocietyofHematology–AmosMedicalFacultyDevelop-
ment program. L.H. is supported by the Swedish Research Council.
S.S.W. is the T. John Gribble Chair of Pediatric Hematology/
Oncology at the University of New Mexico. S.P.H. is the Ergen
Family Chair in Pediatric Cancer at The Children’s Hospital,
Aurora, CO. S.Z. is supported by the Leukemia & Lymphoma
Society, the St Baldrick Foundation, and the Johns M. Driscoll Jr
Children’s Fund. C.G.M. is a Pew Scholar in the Biomedical
Sciences. The array CGH analyses were performed and supported
in part by the Belfer Institute forApplied Cancer Science.
Figure 1. BCL11B inactivation in human T-ALL. (A) Array CGH was performed on genomic DNA from diagnostic lymphoblast specimens collected from 47 children with
T-ALL. The CGH data are shown as a dChip plot of segmented log2copy number ratios. Heterozygous deletions involving BCL11B were identified in 6% (n ? 3 of 47) primary
T-ALL specimens. (B) Sequencing of BCL11B coding sequence was performed in 43 of these cases together with an additional cohort of 70 T-ALL specimens, revealing
heterozygous missense BCL11B mutations in 7 primary patient samples, as well as in 19% (n ? 3 of 16) of T-ALL cell lines. Taken together, we thus identified monoallelic
BCL11B lesions in 9% (n ? 10 of 117) of primary T-ALL patient samples. Mutations are shown based on the full-length CCDS9950.1 BCL11B isoform. (C-E) Homology
structural modeling of canonical DNAbinding by the BCL11B ZF2-ZF3 zinc fingers was performed based on the high-resolution crystal structure of Zif268 in complex with DNA
oligonucleotide.20(C) His445 and His479 are required for coordination of the structural zinc ions of the ZF2 and ZF3 domains, respectively, and their mutations to Tyr are
predicted to unfold of the BCL11B zinc fingers. (D-E) Arg447 and Arg472 form salt bridge and hydrogen bonds with the phosphate backbone and nucleotide bases of DNA,
respectively, and their mutations to His are predicted to disrupt DNAbinding. Yellow dotted lines depict salt bridge and hydrogen bonds.
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Authorship
Contribution: A.G. designed, performed and analyzed research,
and wrote the paper; A.K. designed, performed, and analyzed
research; T.S. performed and analyzed research; J.Z., A.P., and
L.C. developed vital CGH analytical tools and analyzed data;
S.E.D. and D.S.N. analyzed data; L.B.S., S.W., S.P.H., and
S.E.S. provided samples and analyzed data; S.Z. and F.W.A.
designed research and analyzed data; L.H., S.-C.C., J.R.D., and
C.G.M. provided samples, designed and performed research,
and analyzed data; and A.T.L. supervised research, analyzed
data, and cowrote the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Dr A. Thomas Look, Department of Pediatric
Oncology, Dana-Farber Cancer Institute, Mayer 630, 44 Binney St,
Boston, MA02215; e-mail: thomas_look@dfci.harvard.edu.
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