To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML.

Acute myeloid leukemia (AML) will occur in approximately 13 000 new cases per year in the United States and result in 9000 untimely deaths. Recurrent chromosomal alterations provided some of the first clues to the pathogenesis of AML and contributed to improved classification of its subtypes. The molecular characterization of these translocations, along with identification of genes harboring intragenic mutations, has further elucidated disease mechanisms and provided new diagnostic and therapeutic approaches.1,2  These genes include FLT3, NPM1, CEBPA, TP53, IDH1 and IDH2, TET2, WT1, RUNX1, ASXL1, EZH2, NF1, and DNMT3A.2-18  Historically, most mutated genes driving cancers have been thought to regulate specific, well-defined pathways, such as those associated with RAS/RAF, PTEN/PIK3CA, and APC/β-catenin.19  More recently, unbiased genome-wide studies have identified mutated genes that appear to deregulate global patterns of gene expression through effects on chromatin structure or DNA methylation (eg, IDH1/2, TET2, EZH2, and DNMT3A).20-24 

To extend our knowledge of subtle genetic alterations involved in AML, we have studied 8 secondary AML cases by exomic sequencing and have extended findings into a well-characterized unselected cohort of 173 AML cases as well as 19 AML cell lines. Through these efforts, we have identified novel somatically acquired inactivating mutations in the transcriptional corepressor gene BCORL1 in AML.

Patients and samples

The AML patients studied in this report were consecutively enrolled between March 2005 and October 2010 at the University of Michigan Comprehensive Cancer Center. The study was approved by the University of Michigan Institutional Review Board (IRBMED #2004-1022), and written informed consent was obtained from all patients before enrollment in accordance with the Declaration of Helsinki. Clinical and other characteristics of the 8 secondary AML cases used in the discovery screen are detailed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). For the prevalence screen, 165 additional unselected AML cases (exclusive of AML-FAB-M3) were analyzed; the clinical characteristics of this AML cohort are detailed in supplemental Table 2. (For all other methods, see supplemental Methods.)

Patient characteristics

Characteristics of the 173 AML patients (all non-M3 French-American-British subtype) analyzed in this study are summarized in supplemental Tables 1 to 3. Of these patients, 58% had de novo AML, 33% had AML with myelodysplasia-related changes (World Health Organization 2008 MRC1-3), and 9% had treatment-related AML (the latter category does not include patients that received MDS-directed therapies only). Eighty percent of patients had not been treated for AML at trial enrollment, whereas 20% had been treated for AML and relapsed at the time their samples were obtained.

Discovery screen

We determined the sequences of approximately 18 000 protein-encoding genes in 8 patients with secondary AML (supplemental Table 1). Massively parallel sequencing of captured tumor and matched normal DNA resulted in an average depth of coverage of each base in the targeted regions of 92-fold; 94.1% of the bases were represented by at least 10 reads (supplemental Table 4).

Using stringent criteria, we found 5 genes to be somatically mutated in 2 tumors in the discovery set, including BCORL1, NRAS, IDH1, DNMT3A, and RUNX1. We also identified one IDH2 mutation and one TP53 mutation in 1 of 8 discovery tumors.

Prevalence screen

Based on the results of the discovery screen, we initially evaluated the BCORL1, NRAS, IDH1, IDH2, DNMT3A, RUNX1, TP53, FLT3, and NPM1 genes for mutations in an additional 87 AML cases that were intentionally enriched for secondary AML (AML with myelodysplasia-related changes; supplemental Table 5). In each case, we amplified each exon of each gene from purified blast cell DNA and used conventional Sanger sequencing to search for mutations. Any mutations identified in the tumors were tested to determine whether they were somatic by similar sequencing of paired normal DNA from the same patients. Through these efforts, we identified 6 additional cases of AML with BCORL1 mutations. Patient AML 35 was preceded by CMML1. We tested 2 consecutively procured bone marrow samples from patient 35 in the CMML1 phase of the illness and detected the BCORL1 mutation at both times.

In addition to BCORL1, several of the other tested genes had mutations in these additional 87 cases (supplemental Tables 6 and 7). Whereas mutations in FLT3 and NPM1 were more prevalent in primary compared with secondary AML, mutations in BCORL1, DNMT3A, RUNX1, IDH1, and IDH2 were equally distributed. As expected, TP53 mutations were more common in secondary and t-AML than in de novo AML.

Next, we evaluated the BCORL1 gene in an additional 78 AML cases, thus complementing the analysis for this gene for all AML in our consecutively enrolled AML cohort (N = 173). Two additional BCORL1 mutated cases were identified, bringing the total to 10 (of 173 cases analyzed) and providing a BCORL1 mutation prevalence estimate of 5.8%. Characteristics of the 10 AML cases with BCORL1 mutations are summarized in Table 1. None of the BCORL1 mutated cases was TP53, CEBPA, or NPM1 mutated.

Table 1

Characteristics of AML cases with BCORL1 mutations

SampleAge, ySexPrevious treatment for AMLFAB subclassWHO typePrior MDS or MDS/MPNTherapy-relatedSWOG risk groupCytogenetic riskBCORL1 mutationsKRAS mutationsNRAS mutationsCEBPA mutationsIDH1 mutationsIDH2 mutationsDNMT3A mutationsTP53 mutationsRUNX1 mutationsFLT3 mutationsNPM1 mutations
AML14 70 Female No M1 AML-NOS No No Intermediate 47,XX,+8[19] p.S1158A WT WT WT WT p.R140Q WT WT Frameshift p.D835Y WT 
AML151 51 Male No M4 AML-MRC-1,2 RAEB-1 No Unfavorable 45,XY,-7,i(21)(q10)[12]/46,XY, +i(21)(q10)[6] p.P810T WT p.Q61K WT WT WT WT WT p.W106L WT WT 
AML33 68 Male No M6 AML-MRC-1 RAEB-2 No Intermediate 46,XY,del(3)(q13q27)[16]/46,XY[4] Frameshift WT WT WT WT WT p.V716I WT WT WT WT 
AML35 67 Male No M2 AML-MRC-1 CMML-1 No Intermediate 47,XY,+14[16]/46,XY[4] Frameshift WT WT WT WT WT p.Y448X WT WT WT WT 
AML45 63 Female No M4 AML-TR No Yes Intermediate 46,XY[20] Frameshift WT WT WT WT WT WT WT WT WT WT 
AML216 50 Female No M5 AML-MRC-1 RCMD No Intermediate 47,XX,+8[5]/46,XX[24] Frameshift WT WT WT p.R132C WT p.S714F, p.G685R WT WT WT WT 
AML257 63 Female No M4 AML-NOS No No Intermediate 46,XX[20] Frameshift WT WT WT WT WT WT WT Frameshift; p.F131V ITD WT 
AML57 69 Male No M4eo AML-RGA (16p13q22) No No Favorable 46,XY,inv(16)(p13q22)[14] p.Q538X p.G13GD WT WT WT WT WT WT WT WT WT 
AML87 55 Female No M0 AML-NOS No No Unfavorable 46,XX,t(3;9;22)(q21;q34;q11.2) [15]/46,XX[5] p.E1112X WT WT WT WT WT WT WT WT WT WT 
AML182 59 Male No M0 AML-NOS No No Intermediate 48,XY,+21,+21[15] p.R784X WT WT WT p.R132G WT p.R693H WT Frameshift ITD WT 
SampleAge, ySexPrevious treatment for AMLFAB subclassWHO typePrior MDS or MDS/MPNTherapy-relatedSWOG risk groupCytogenetic riskBCORL1 mutationsKRAS mutationsNRAS mutationsCEBPA mutationsIDH1 mutationsIDH2 mutationsDNMT3A mutationsTP53 mutationsRUNX1 mutationsFLT3 mutationsNPM1 mutations
AML14 70 Female No M1 AML-NOS No No Intermediate 47,XX,+8[19] p.S1158A WT WT WT WT p.R140Q WT WT Frameshift p.D835Y WT 
AML151 51 Male No M4 AML-MRC-1,2 RAEB-1 No Unfavorable 45,XY,-7,i(21)(q10)[12]/46,XY, +i(21)(q10)[6] p.P810T WT p.Q61K WT WT WT WT WT p.W106L WT WT 
AML33 68 Male No M6 AML-MRC-1 RAEB-2 No Intermediate 46,XY,del(3)(q13q27)[16]/46,XY[4] Frameshift WT WT WT WT WT p.V716I WT WT WT WT 
AML35 67 Male No M2 AML-MRC-1 CMML-1 No Intermediate 47,XY,+14[16]/46,XY[4] Frameshift WT WT WT WT WT p.Y448X WT WT WT WT 
AML45 63 Female No M4 AML-TR No Yes Intermediate 46,XY[20] Frameshift WT WT WT WT WT WT WT WT WT WT 
AML216 50 Female No M5 AML-MRC-1 RCMD No Intermediate 47,XX,+8[5]/46,XX[24] Frameshift WT WT WT p.R132C WT p.S714F, p.G685R WT WT WT WT 
AML257 63 Female No M4 AML-NOS No No Intermediate 46,XX[20] Frameshift WT WT WT WT WT WT WT Frameshift; p.F131V ITD WT 
AML57 69 Male No M4eo AML-RGA (16p13q22) No No Favorable 46,XY,inv(16)(p13q22)[14] p.Q538X p.G13GD WT WT WT WT WT WT WT WT WT 
AML87 55 Female No M0 AML-NOS No No Unfavorable 46,XX,t(3;9;22)(q21;q34;q11.2) [15]/46,XX[5] p.E1112X WT WT WT WT WT WT WT WT WT WT 
AML182 59 Male No M0 AML-NOS No No Intermediate 48,XY,+21,+21[15] p.R784X WT WT WT p.R132G WT p.R693H WT Frameshift ITD WT 

FAB indicates French-American-British; WHO, World Health Organization; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; SWOG, Southwest Oncology Group; NOS, not otherwise specified; MRC, Medical Research Council; RAEB, refractory anemia with excess blasts; CMML, chronic myelomonocytic leukemia; RCMD, refractory cytopenia with multilineage dysplasia; and WT, wild-type.

The coding exons of the BCORL1 gene were also sequenced in 24 cell lines, of which 19 were AML-derived. In total, 5 BCORL1 sequence variants were identified, including 3 nonsense, 1 frameshift, and 1 splice junction mutation (located in the conserved nucleotide immediately preceding exon 10). Data are summarized in supplemental Table 8.

BCORL1 is a transcriptional corepressor encoding a protein that is found tethered to promoter regions by DNA-binding proteins. BCORL1 contains a putative bipartite nuclear localization signal, tandem ankyrin repeats, and a classic C-terminal binding protein (CtBP)–binding motif, PXDLS. Functional studies25  have shown that BCORL1 can interact with class II histone deacetylases, interact with the CtBP corepressor through the CtBP binding motif-PXDLS and affects the repression of E-cadherin.25 

The BCORL1 gene is located on the X-chromosome at cytoband Xq25-q26.1 at approximately physical position 129.14 to 129.19 Mb. Eight of 10 (80%) BCORL1 mutations found in primary cells and all BCORL1 mutations in cell lines truncated the encoded protein as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. All the truncating mutations in BCORL1 were predicted to result in severely shortened polypeptides lacking the LXXLL nuclear receptor recruitment motif and the C-terminus (Figure 1). These data not only provide strong evidence that BCORL1 mutations are important to the pathogenesis of AML but demonstrate that BCORL1 is, on the basis of genetic criteria, a tumor suppressor gene that is inactivated by mutations.

Figure 1

Schematic representations of the location of somatic mutations identified in the BCORL1 gene. (A) Examples of sequence chromatograms showing inactivating somatic mutations of BCORL1 in blast cells (bottom panels) but not in their matched normal buccal mucosa (top panels). (B) Schematic of somatic mutations identified in the BCORL1 gene in primary AML cells and AML cell lines. Red arrows indicate truncating mutations; and black arrows, missense mutations. NLS indicates nuclear localization signal; LXXLL, Leu-Xaa-Xaa-Leu-Leu motif; ANK, tandem ankyrin repeats; aa, amino acids; and CtBP, C-terminal binding protein.

Figure 1

Schematic representations of the location of somatic mutations identified in the BCORL1 gene. (A) Examples of sequence chromatograms showing inactivating somatic mutations of BCORL1 in blast cells (bottom panels) but not in their matched normal buccal mucosa (top panels). (B) Schematic of somatic mutations identified in the BCORL1 gene in primary AML cells and AML cell lines. Red arrows indicate truncating mutations; and black arrows, missense mutations. NLS indicates nuclear localization signal; LXXLL, Leu-Xaa-Xaa-Leu-Leu motif; ANK, tandem ankyrin repeats; aa, amino acids; and CtBP, C-terminal binding protein.

We also measured normalized mRNA levels for BCORL1 in cDNA made from RNA isolated from FACS-sorted AML blasts in an expanded cohort of AML cases. In summary, we found relatively uniform expression of BCORL1 and all studied cases expressed the gene, including those with BCORL1 mutations (supplemental Table 9).

Analysis of the BCORL1 gene status in the AML cohort using SNP 6.0 array profiling uncovered no microdeletions or gains spanning the gene. Furthermore, copy-neutral loss of heterozygosity was not identified (supplemental Figure 1).

DNMT3A mutations have recently been reported in 22.1% (62 of 281) of de novo AML5  and in 20.5% of predominantly new diagnosed AML.6  Similarly, somatic mutations in DNMT3A were identified in 23.2% (22 of 95) of the cases we analyzed (supplemental Table 10). The novel DNMT3A-related finding in our study was a frameshift mutation in normal buccal mucosa and in remission marrow and neoplastic cells of AML patient89 (supplemental Figure 2). No additional mutations of DNMT3A were identified in the patient's tumor; the patient had no other tumors and was 78 years old. These data raise the possibility that germline DNMT3A mutations can weakly predispose persons to the development of AML but not to other tumors. There was no history of AML in this patient's family, and the parents were unavailable to determine whether the mutation was de novo.

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.

This work was supported by the Leukemia & Lymphoma Society (Clinical Scholar Award, S.N.M.), Virginia and D. K. Ludwig Fund for Cancer Research, and National Institutes of Health (grants CA46592, CA43460, and CA57345).

National Institutes of Health

Contribution: M.L., B.V., and S.N.M. conceived the study and supervised the work; M.L., P.O., Y.J., S.N.M., N.P., K.W.K., and B.V. performed the laboratory research and genomic data analysis; R.C., H.E., D.B., and S.N.M. enrolled patients and contributed and analyzed clinical data; and M.L., B.V., and S.N.M. wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Nickolas Papadopoulos, Ludwig Center for Cancer Genetics and Therapeutics and Howard Hughes Medical Institute, Johns Hopkins Kimmel Cancer Center, 401 N Broadway, Baltimore, MD 21231; e-mail: npapado1@jhem.jhmi.edu; and Sami N. Malek, Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, 4312 Cancer Center, 1500 E Medical Center Dr, Ann Arbor, MI 48109; e-mail: smalek@med.umich.edu.

1
Lowenberg
 
B
Acute myeloid leukemia: the challenge of capturing disease variety.
Hematology Am Soc Hematol Educ Program
2008
(pg. 
1
-
11
)
2
Breems
 
DA
Van Putten
 
WL
De Greef
 
GE
, et al. 
Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype.
J Clin Oncol
2008
, vol. 
26
 
29
(pg. 
4791
-
4797
)
3
Grimwade
 
D
Hills
 
RK
Moorman
 
AV
, et al. 
Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials.
Blood
2010
, vol. 
116
 
3
(pg. 
354
-
365
)
4
Schlenk
 
RF
Dohner
 
K
Krauter
 
J
, et al. 
Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia.
N Engl J Med
2008
, vol. 
358
 
18
(pg. 
1909
-
1918
)
5
Ley
 
TJ
Ding
 
L
Walter
 
MJ
, et al. 
DNMT3A mutations in acute myeloid leukemia.
N Engl J Med
2010
, vol. 
363
 
25
(pg. 
2424
-
2433
)
6
Yan
 
XJ
Xu
 
J
Gu
 
ZH
, et al. 
Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia.
Nat Genet
2011
, vol. 
43
 
4
(pg. 
309
-
315
)
7
Mardis
 
ER
Ding
 
L
Dooling
 
DJ
, et al. 
Recurring mutations found by sequencing an acute myeloid leukemia genome.
N Engl J Med
2009
, vol. 
361
 
11
(pg. 
1058
-
1066
)
8
Nikoloski
 
G
Langemeijer
 
SM
Kuiper
 
RP
, et al. 
Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes.
Nat Genet
2010
, vol. 
42
 
8
(pg. 
665
-
667
)
9
Ernst
 
T
Chase
 
AJ
Score
 
J
, et al. 
Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders.
Nat Genet
2010
, vol. 
42
 
8
(pg. 
722
-
726
)
10
Dufour
 
A
Schneider
 
F
Metzeler
 
KH
, et al. 
Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome.
J Clin Oncol
2010
, vol. 
28
 
4
(pg. 
570
-
577
)
11
Delhommeau
 
F
Dupont
 
S
Della Valle
 
V
, et al. 
Mutation in TET2 in myeloid cancers.
N Engl J Med
2009
, vol. 
360
 
22
(pg. 
2289
-
2301
)
12
Hou
 
HA
Huang
 
TC
Lin
 
LI
, et al. 
WT1 mutation in 470 adult patients with acute myeloid leukemia: stability during disease evolution and implication of its incorporation into a survival scoring system.
Blood
2010
, vol. 
115
 
25
(pg. 
5222
-
5231
)
13
Schnittger
 
S
Dicker
 
F
Kern
 
W
, et al. 
RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis.
Blood
2011
, vol. 
117
 
8
(pg. 
2348
-
2357
)
14
Tang
 
JL
Hou
 
HA
Chen
 
CY
, et al. 
AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations.
Blood
2009
, vol. 
114
 
26
(pg. 
5352
-
5361
)
15
Virappane
 
P
Gale
 
R
Hills
 
R
, et al. 
Mutation of the Wilms' tumor 1 gene is a poor prognostic factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: the United Kingdom Medical Research Council Adult Leukaemia Working Party.
J Clin Oncol
2008
, vol. 
26
 
33
(pg. 
5429
-
5435
)
16
Thiede
 
C
Koch
 
S
Creutzig
 
E
, et al. 
Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).
Blood
2006
, vol. 
107
 
10
(pg. 
4011
-
4020
)
17
Grimwade
 
D
Hills
 
RK
Independent prognostic factors for AML outcome.
Hematology Am Soc Hematol Educ Program
2009
(pg. 
385
-
395
)
18
Abdel-Wahab
 
O
Mullally
 
A
Hedvat
 
C
, et al. 
Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies.
Blood
2009
, vol. 
114
 
1
(pg. 
144
-
147
)
19
Vogelstein
 
B
Kinzler
 
KW
Cancer genes and the pathways they control.
Nat Med
2004
, vol. 
10
 
8
(pg. 
789
-
799
)
20
Ko
 
M
Huang
 
Y
Jankowska
 
AM
, et al. 
Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.
Nature
2010
, vol. 
468
 
7325
(pg. 
839
-
843
)
21
Ward
 
PS
Patel
 
J
Wise
 
DR
, et al. 
The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.
Cancer Cell
2010
, vol. 
17
 
3
(pg. 
225
-
234
)
22
Ito
 
S
D'Alessio
 
AC
Taranova
 
OV
Hong
 
K
Sowers
 
LC
Zhang
 
Y
Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.
Nature
2010
, vol. 
466
 
7310
(pg. 
1129
-
1133
)
23
Figueroa
 
ME
Abdel-Wahab
 
O
Lu
 
C
, et al. 
Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation.
Cancer Cell
2010
, vol. 
18
 
6
(pg. 
553
-
567
)
24
Jiang
 
Y
Dunbar
 
A
Gondek
 
LP
, et al. 
Aberrant DNA methylation is a dominant mechanism in MDS progression to AML.
Blood
2009
, vol. 
113
 
6
(pg. 
1315
-
1325
)
25
Pagan
 
JK
Arnold
 
J
Hanchard
 
KJ
, et al. 
A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP.
J Biol Chem
2007
, vol. 
282
 
20
(pg. 
15248
-
15257
)

Supplemental data