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Regular Article
MYELOID NEOPLASIA
Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD
at diagnosis and relapse
Manoj Garg,1 Yasunobu Nagata,2 Deepika Kanojia,1 Anand Mayakonda,1 Kenichi Yoshida,2 Sreya Haridas Keloth,1
Zhi Jiang Zang,1 Yusuke Okuno,3 Yuichi Shiraishi,4 Kenichi Chiba,4 Hiroko Tanaka,5 Satoru Miyano,5 Ling-Wen Ding,1
Tamara Alpermann,6 Qiao-Yang Sun,1 De-Chen Lin,1 Wenwen Chien,1 Vikas Madan,1 Li-Zhen Liu,1 Kar-Tong Tan,1
Abhishek Sampath,1 Subhashree Venkatesan,1 Koiti Inokuchi,7 Satoshi Wakita,7 Hiroki Yamaguchi,7 Wee Joo Chng,1
Shirley-Kow Yin Kham,8 Allen Eng-Juh Yeoh,8 Masashi Sanada,2,9 Joanna Schiller,10 Karl-Anton Kreuzer,10
Steven M. Kornblau,11,12 Hagop M. Kantarjian,11,12 Torsten Haferlach,6 Michael Lill,13 Ming-Chung Kuo,14 Lee-Yung Shih,14
Igor-Wolfgang Blau,15 Olga Blau,15 Henry Yang,1 Seishi Ogawa,2 and H. Phillip Koeffler1,13,16
1
Cancer Science Institute of Singapore, National University of Singapore, Singapore; 2Department of Pathology and Tumor Biology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan; 3Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan; 4Laboratory of DNA
Information Analysis, and 5Laboratory of Sequence Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
6
Munich Leukemia Laboratory, Munich, Germany; 7Department of Hematology, Nippon Medical School, Tokyo, Japan; 8Department of Paediatrics, National
University Health System, Singapore; 9Department of Advanced Diagnosis, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan;
10
Department I of Internal Medicine, University of Cologne, Cologne, Germany; 11Department of Leukemia, MD Anderson Cancer Center, Houston, TX;
12
Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX; 13CedarsSinai Medical Center, Division of Hematology/Oncology, University of California Los Angeles, School of Medicine, Los Angeles, CA; 14Division of
Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan; 15Department of
Hematology, Oncology and Tumorimmunology, Charite University School of Medicine, Berlin, Germany; and 16National University Cancer Institute, National
University Hospital, Singapore
Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD)
mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the
mutational spectrum associated with relapse, whole-exome sequencing was performed on
• MLL3 acts as tumor
suppressor in FLT3-ITD AML. 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299
genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per
• The existence of DNMT3A
sample, similar to other AML subtypes, which is a low mutation rate compared with that in
mutations in remission
solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin,
samples implies that the
histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex,
DNMT3A mutant clone can
and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated
survive induction
genes suggests that these genes have a strong biological relationship. In addition, we
chemotherapy.
identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4,
and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A
mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions.
Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral
clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from
induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in
transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype.
An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that
FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone. (Blood. 2015;126(22):2491-2501)
Key Points
Introduction
Acute myelogenous leukemia (AML) is a clonal disorder of hematopoietic stem and progenitor cells caused by acquired and occasionally
inherited genetic alterations.1 Fms-like tyrosine kinase 3 (FLT3) is a
tyrosine kinase receptor involved in proliferation and differentiation
of hematopoietic stem cells. Constitutive activation of FLT3 by internal tandem duplication (ITD) mutation is one of the most common
molecular alterations in AML, occurring in approximately 20% to 30%
of AML patients who have a comparatively poor clinical outcome and
increased relapse rate.1-3 This oncogenic mutation inappropriately
activates the cell surface tyrosine kinase receptor to signal downstream
pathways via mitogen-activated protein kinase/extracellular signalregulated kinase and phosphatidylinositol-3 kinase/AKT pathways.
Submitted May 18, 2015; accepted September 22, 2015. Prepublished online
as Blood First Edition paper, October 5, 2015; DOI 10.1182/blood-2015-05646240.
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.
The online version of this article contains a data supplement.
© 2015 by The American Society of Hematology
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GARG et al
FLT3-ITD also potently activates STAT5 pathway-stimulating transcription of target genes (eg, cyclin-D1, c-Myc, and antiapoptotic genes
p21 and Bcl-XL).4-7 Thus, FLT3-ITD enhances cellular proliferation
and reduces apoptosis of hematopoietic blasts.
Although 70% of FLT3-ITD AML patients achieve a complete remission (CR) with conventional chemotherapy, the majority of patients
eventually relapse and die of therapy-resistant leukemia.8 Recently,
several small-molecule tyrosine kinase inhibitors targeting FLT3 have
been investigated in phase 1/2 clinical trials.9-12 FLT3 inhibitors are
efficient for a limited period of time in treating AML patients because
these compounds are well tolerated at doses that achieve inhibition of
FLT3.13,14 Heterozygous FLT3WT/ITD knockin mice developed a myeloproliferative neoplasm. However, these mice did not develop acute
leukemia, suggesting that additional drivers are required for leukemogenesis.15,16 The additional mutations that cause AML include
AML1-ETO fusion gene,17 CBFb-SMMHC gene fusion,18 NUP98
translocations,19 NPM1,20 and loss of Tet2.21 Recently, many studies
have reported exome sequencing of AML, but these studies have a
limited number of FLT3-ITD subtypes and almost none have matched
diagnosis and relapse samples.22-27 Therefore, a need exists to identify
genomic abnormalities underlying the FLT3-ITD subtype at diagnosis
(DX) and at relapse (REL) for a greater understanding of this disease
and to guide the development of effective targeted therapies.
Clustering and clonal analysis, mutational signature, frequency analysis
using TDS, significantly mutated gene analysis, pathway analysis, single
nucleotide polymorphism (SNP) array analysis,31,32 cell culture, antibodies,
RNA interference, reverse transcription polymerase chain reaction (PCR) analysis and quantitative real-time PCR,33 gel electrophoresis and immunoblotting,33
and xenograft models33 are described in the supplemental Data.
Short-term cell proliferation assay
MOLM14 and MV4-11cells were transfected twice with scramble mixedlineage leukemia 3 (MLL3) or FAT1 small interfering RNA (siRNA). The second
transfection was performed after an additional 48 hours. After the second
transfection, 8,000 cells were seeded in 96-well plates, and cell proliferation was
measured by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (Sigma-Aldrich). Cells were incubated with 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (0.5 mg/mL) and incubated for
3 hours in a CO2 incubator at 37°C. Formazan crystals were dissolved in 100 mL
of stop solution (sodium dodecyl sulfate HCl). Absorbance was measured at
570 nm by using a Tecan Infinite 200 PRO spectrophotometer (Tecan,
Mannedorf, Switzerland).
Colony-forming assay
For clonogenic assay, MV4-11 cells were harvested and counted after their
second transfection with siRNA against MLL3, FAT1, or scramble MLL3, and
500 cells per condition were plated in Methocult H4230 medium (STEMCELL
Technologies) and cultured for 7 to 10 days at 37°C in a humidified 5% CO2
incubator. Colonies consisting of more than 50 cells were counted in both the
control and experimental wells.
Methods
Statistical analysis
Patients and samples
Genomic DNAs (gDNAs) from 80 AML patient samples with the FLT3-ITD
subgroup were collected at 3 different time points (DX, CR, and REL) by
collaborating with several institutes. The German cohort consisted of 56 patients
(38 with DX, CR, and REL samples and 18 with DX and CR samples) provided
by Charite University School of Medicine, Berlin and Munich Leukemia
Laboratory, Munich, Germany. The Japanese samples (3 patients with DX, CR,
and REL samples) were provided by Nippon Medical School, Tokyo, Japan. The
Taiwanese cohort consisted of 12 patients (7 with DX, CR, and REL samples and
5 with CR and REL samples) provided by Chang Gung Memorial Hospital,
Taipei, Taiwan. The Singaporean cohort consisted of 9 patients (2 with DX, CR,
and REL samples and 7 with DX and CR samples) provided by the National
University Health System, Singapore. All samples were collected between
September 1997 and August 2012. These samples were used for either wholeexome sequencing (WES) or targeted deep sequencing (TDS). Written independent consent was obtained from all patients for research studies using their
samples. Clinical characteristics of these patients included age and sex, the distribution of French-American-British subtype, cytogenetics, bone marrow white
blood cell counts, hemoglobin, platelets, survival, and FLT3-ITD status (supplemental Tables 1 and 6 available on the Blood Web site). Patients were treated with
standard chemotherapy, including induction chemotherapy (100 mg/m2 cytosine
arabinoside on days 1 to 7 via continuous intravenous dosing and 60 mg/m2
daunorubicin or idarubicin on days 4 to 6). Patients who achieved CR were
usually given consolidation chemotherapy that involved either the same or
similar drugs. FLT3-ITD AML patients with primary refractory disease were
not included in this study. gDNA was extracted by using a QIAamp DNA
Blood Mini Kit and a QIAamp DNA Investigator kit (QIAGEN). The Qubit
dsDNA BR Assay Kit (Life Technologies) was used to quantify the
concentration of gDNA. The intactness of each gDNA sample was assessed
by agarose gel electrophoresis using 0.7% agarose gel.
Exome capture, massively parallel sequencing, and analysis of
WES data
gDNAs from DX, CR, and REL samples were used for WES as described
previously28-30 (details are provided in the supplemental Data).
The false discovery rate q values for mutated genes were calculated by using
the Mutational Significance in Cancer (MuSiC; http://gmt.genome.wustl.edu/
packages/genome-music/) suite of tools with default parameters. The statistical
analyses were conducted by using the two-tailed Student t test upon verification
of the assumptions (eg, normality); otherwise, the nonparametric test was applied
for short-term cell proliferation, quantitative PCR, colony formation, and
xenografts.
Results
Identifying somatic mutations by using WES and TDS
Initially, WES was performed on 13 trios consisting of samples at DX
and REL along with their CR (germ line control) as the discovery
cohort. The average coverage of each base in the targeted region was
120-fold, with 78% of bases covered at least 20X (supplemental Tables
1 and 2 and supplemental Figure 1). SNP array (copy number analysis)
was performed on 11 of these 13 trio samples. To eliminate contamination of the leukemic cells in CR, we included only those samples
whose bone marrow blast counts in CR were ,5% (supplemental
Table 1).
In total, 179 nonsilent somatic mutations of the WES of the discovery cohort were confirmed by Sanger sequencing, including 131
missense, 8 nonsense, 5 splice site mutations, 22 indels, and 13 in-frame
insertion mutations (true positive rate, 92.1%; supplemental Tables 3
and 4 and supplemental Figure 2A-B). The average number of validated
somatic mutations per sample was 13.6 (Figure 1A and supplemental
Table 3), a mutation rate comparable to that in most hematologic
malignancies23-27,30,34 but significantly lower than that present in solid
tumors.35-40 A total of 75 shared mutations occurred at both DX and
REL. Moreover, 64 mutations were DX specific, and 26 mutations were
REL specific (supplemental Figure 2C).
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Figure 1. Somatic mutations identified by WES and TDS of FLT3-ITD AML. (A) Number of mutations discovered for 50 individuals at DX, CR, and REL (light gray), 25
individuals at DX and CR (dark gray), and 5 individuals at CR and REL (black). Samples subjected to WES plus TDS or TDS only are color coded in light or dark brown,
respectively. Dark blue, red, and dark green represent the number of somatic mutations at DX, REL, and both DX and REL, respectively. Light blue and magenta show the
number of mutations observed in DX and REL samples, respectively. (B) Overall frequency of mutated genes in 80 FLT3-ITD AML patients. NPM1 gene mutations are
missense frameshift mutations (green). (C) Frequency of specific somatic mutations detected only at REL in 50 trios (DX, CR, and REL paired).
TDS of 151 genes (ascertained from the discovery cohort plus an
additional 148 genes related to hematologic malignancies [The Cancer
Genome Atlas [TCGA], http://cancergenome.nih.gov/; Cancer Gene
Census, http://cancer.sanger.ac.uk/cancergenome/projects/census/]
supplemental Table 5) was performed on 67 additional FLT3-ITD patient samples (supplemental Table 6), as well as 2 FLT3-ITD-positive
AML cell lines (frequency cohort). We also included 13 patients from
the discovery cohort to ensure capture efficiency. Samples in the frequency cohort had a mean average coverage of the targeted regions of
107-fold, with 77% of the bases covered at least 30X (supplemental
Table 7 and supplemental Figure 3). In total, 284 nonsilent somatic
mutations were verified by Sanger sequencing with an average of 4.23
mutations per sample (true positive rate, 91.03%; Figure 1A and supplemental Tables 8 and 9). A total of 42 genes were mutated in more
than 2 samples (supplemental Table 8), with 21 genes mutated at a
frequency of more than 4% (Figure 1B). Recurrent mutations in 9 genes
were also identified in the REL-specific phase (Figure 1C). WES and
TDS found a high frequency of mutations of the NPM1 gene (36%), as
well as genes related to DNA methylation (63%), chromatin modification (42%), histone methylation (38%), myeloid transcription factors
(22%), tumor suppressors (20%), signaling (16%), adhesion (14%),
lipid metabolism (11%), cohesin complex (10%), and spliceosomes
(10%) (Figure 2). Hotspot mutations also occurred in several genes:
DNMT3A (R882H and R882C), NPM1 (W288fs), FLT3-TKD (D835Y,
D839G, Y597F, L601F, and N676), SF3B1 (K666N and K700E),
U2AF1 (S34F and S34Y), IDH1 (R132H and R132C), and IDH2
(R140Q) (supplemental Figures 4A-C and 5A-D).
Heterozygous DNMT3A mutations were confirmed in 19 of 50 trios.
The same DNMT3A mutation occurred at both DX and REL in 16
(85%) of 19 trios samples, whereas in 2 patients, DNMT3A mutations
were lost at REL, and 1 patient with wild-type DNMT3A at DX gained a
DNMT3A mutation at REL (supplemental Figure 6). Of note, DNMT3A
mutations were detected at a variant allele frequency of ,5% to 40%
in 8 of 19 CR samples (supplemental Figure 7 and supplemental
Table 10). This suggests that use of CR samples as a germline control
limits the power to detect the mutations that exist in early clones that
persist during CR. NPM1 mutations were lost in 3 of 22 patients at REL,
whereas 19 of 22 patients had the same NPM1 mutation at both DX and
REL (supplemental Figures 6 and 8).
The ITD mutations of the FLT3 locus were detected by using
Pindel41 and Genomon ITDetector,42 which has high sensitivity to
detect ITDs using soft clipped reads. All ITDs were detected in exon
14 of the FLT3 gene. In a majority of the cases (45 of 50 trios), ITDs
occurred at the same position and with the same insertion sequence at
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Figure 2. Distribution of somatic mutations in 80 patients with FLT3-ITD AML. Each column displays French-American-British classification, sex, and ethnicity of an
individual sample; each row denotes a specific gene. Recurrently mutated genes are color coded for missense (blue), nonsense (red), indel (green), splice site (purple), and
stoploss (gray). The letters D and R or diagonal lines denote somatic mutation at DX, REL, and both DX and REL, respectively. Asterisks mark genes mutated at a significant
(false discovery rate ,0.05) recurrence rate. Mutated genes are clustered according to their pathways or family.
both the DX and REL stages of the disease. However, 5 patients showed
different ITD insertion at REL (supplemental Figure 6). On the basis of
calculated variant allele frequency, FLT3-ITD was present in subclones
at both DX and REL in 92% of the cases (46 of 50). However,
FLT3-ITD was also observed as a part of the founder clone at DX and
REL in 4% of the cases (2 of 50) (supplemental Table 11).
The MuSiC suite of tools was used to identify significantly mutated
genes or selected candidate driver genes.43,44 Twenty genes with a
higher-than-expected mutational prevalence were identified, including
the well-known drivers relevant to AML pathogenesis (eg, DNMT3A,
NPM1, WT1, FLT3, RUNX1, GATA2, TET2, EZH2, IDH1, and IDH2),
along with novel genes that have only recently been implicated in
AML pathogenesis, including MLL3, KDM6A, FAT1, NSD1, ARID2,
SRCAP, and FAT4 (Figure 2 and supplemental Table 12). By using the
MuSiC PathScan algorithm, 6 pathways were significantly enriched
in the mutated gene list45,46 (supplemental Table 13). Moreover, cooccurrence was observed between mutations in DNMT3A and NPM1,
DNMT3A and FAT1 (P , .032), IDH2 and NPM1 (P , .032), MLL3
and SRCAP (P , .05), EZH2 and KDM6 (P , .001), and FAT1 and
NSD1 (P , .04). Mutual exclusivity was identified between NPM1 and
RUNX1 (P , .036), NPM1 and MLL3 (P , .001), WT1 and MLL3
(P , .04), and MLL3 and RUNX1 (supplemental Figure 9A). Mutual
exclusivity especially occurred in mutations of genes belonging to
different biological categories, such spliceosomes, cohesin complex
(supplemental Figure 9B), chromatin modifiers, and histone-modifying
proteins (supplemental Figure 10A-B), suggesting that 1 mutation in
these pathways is generally adequate for AML pathogenesis.
One of our goals in this study was to identify genomic therapeutic
targets in FLT3-ITD AML. Ten genes with potentially druggable
alterations were noted in 52 (65%) of 80 FLT3-ITD patients (supplemental Figure 11).
Mutational classes and mutational signature in FLT3-ITD AML
Discovery cohort patients were treated with cytarabine and
daunorubicin (anthracycline) for induction therapy (supplemental
Table 1). To examine the possible effects of chemotherapeutic agents
on the mutational spectrum of REL samples, 6 classes of transition
and transversion mutations were compared at DX vs REL.23 The C.T
transitions were the most common mutations at DX and REL in the
AML genomes, but their frequencies were different (P 5 .012)
between DX-specific mutations (56%) and REL-specific mutations
(38%). The average increase in C.A (26%; P 5 .0078) and C.G
transversions occurred in REL-specific mutations (Figure 3A). In
addition, overall frequency of transversions was higher in RELspecific AML samples (Figure 3B).
Each mutational process usually induces a unique combination
of nucleotide changes that provide a signature. Mutational signature
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Figure 3. Distribution of mutational nucleotide classes between DX and REL paired samples. (A) Proportion of nucleotide transition and transversion mutations at DX
and REL of 14 patients studied by WES. (B) Overall frequency of transversions at DX and REL (13 patients). Z test (proportion test) was used for statistical significance. (C-D)
Mutational signature using a 96 substitution classification based on substitution classes and the sequence context immediately to the 59 and 39 ends of the mutated base.
Mutation types are represented using different colors. Horizontal axes display type of mutations; vertical axes represent percentage of mutations in a specific mutation type.
(E) Percentage contribution of the two mutational processes identified by EMu analysis.
analysis using EMu software (https://www.sanger.ac.uk/resources/
software/emu/) showed that signatures 1 and 2 were enriched in the
discovery cohort47-49 (Figure 3C-D). Signature 1 was enriched with C.T
transition at cytosine-phosphate-guanine (CpG) dinucleotides, an intrinsic mutational process reflecting deamination of 5-methylcytosine.
Signature 2 showed predominance of C.T (transition) and C.A and
C.G (transversion) in a thymine-phosphate-cytosine (TpC) context with
enrichment in many REL samples (Figure 3E), which may be induced by
chemotherapy in these REL samples.
Clonal evolution in FLT3-ITD AML
Several forms of clonal evolution occurred. The first is characterized
by patient UPN001 (Figure 4A-C). Mutational clustering at DX and
REL (UPN001) identified 4 clones with well-defined sets of
mutations.23 Median mutant variant allele frequencies (VAFs) of
clones (1 and 2) at DX were 44.4% and 23.53%, respectively. Clone
1 is the founder clone because all of these mutations are heterozygous with VAFs of ;40% to 50%, and they are present in virtually all
the leukemic cells at DX and REL (Figure 4A). Cluster 2 represents
mutations present only in the DX clone. Clusters 3 and 4 were
detected in the REL samples, suggesting that the founder clone
gained additional (REL-specific) mutations, and these mutations
perhaps provided resistance to chemotherapy (Figure 4A-C). Loss of
DX-specific clone at REL suggested that DX-specific clones were
indeed eradicated by chemotherapy (Figure 4C). In this case, NPM1
mutation in REL-specific clones seems to be a driver mutation,
because NPM1 is recurrently mutated in AML. Mutational clustering
of an additional 10 DX-REL pairs revealed 2 different types of clonal
evolution at REL. In type 1 (UPN001 and UPN004), the dominant
clone at DX accumulated more mutations and evolved as an RELspecific clone. In type 2 (UPN002, UPN003, UPN005, UPN007, and
UPN008), a subclone bearing DX-specific mutations escaped from
therapy, acquired additional mutations, and expanded at REL
(Figure 4D-E). Additional mutations in the subclones may provide
resistance to chemotherapy and be an important force for REL
(Figure 4F). In UPN005 and UPN009, DNMT3A mutations were
confirmed in CR samples with VAFs of 20% and 40%, respectively.
This suggests that in these patients, the DNMT3A clone may persist
in morphologic CRs and expand during CR resulting in REL in these
patients. Our survival data also show that these patients have very
short REL-free survival and overall survival.
MLL3 acts as a tumor suppressor gene in FLT3-ITD AML
MLL3 belongs to the TRX/MLL gene family mapping to chromosome
7q36.1,50,51 is reported to be mutated in several types of tumors52-54
(COSMIC, http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/),
and is also deleted in myeloid leukemias.55 Within the TCGA cohort,
only 1 of 200 AML patients had a nonsense mutation (E2793X)
(Figure 5A), and 24 (12%) of 200 AML patients had deletions in the
MLL3 gene24 (supplemental Figure 13A). Interestingly, we observed
somatic mutations of MLL3 in 15% (12 of 80) of FLT3-ITD samples,
including 1 frameshift and 3 stop-gain mutations (Figure 5A). Our
SNP chip data also showed that 3 of 11 samples had deletion of MLL3
(supplemental Figure 12). Interestingly, 2 of the 20 patients with
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Figure 4. Clonal evolution from primary to REL in UPN001 and UPN002 and pattern of evolution observed in 13 DX and REL pairs. (A,D) Distribution of variant allele
frequencies (VAFs) of validated mutations at DX and REL (UPN001 and UPN002). VAFs of genes in region of uniparental disomy are halved. Driver mutations, including
FLT3-ITD, are indicated. Two mutational clusters were identified at DX and 2 were present at REL; 1 was present at both DX and REL. (B,E) Graphic representation of the
relationship between clusters at DX and REL. Gray cluster represents founding clone at DX and REL. (C,F) Schematic representation of mutational clones and their evolution
from DX to REL. Founder clone at DX evolved into REL clones by acquiring REL-specific mutations. HSC, hematopoietic stem cell.
FLT3-ITD in the TCGA cohort also had an MLL3 deletion24
(supplemental Figure 13A).
To study biological consequences of MLL3 inactivation in the
FLT3-ITD subgroup of AML, MV4-11 cells (FLT3-ITD AML)
were transduced with either MLL3 short hairpin RNA (shRNA) or
scramble shRNA. MLL3 silencing resulted in significantly increased cell growth in liquid culture and clonogenic growth in
methylcellulose (Figure 5B-F). Xenograft growth was also significantly greater (size and weight of tumors) compared with
scrambled knockdown cells (Figure 6A-C). To confirm on-target
effects of shRNA knockdown, MLL3 expression was also silenced
with siRNA, and similar results were observed (supplemental
Figure 13B-D). MLL3 depletion in MV4-11 cells resulted in decreased p21 and p53 messenger RNA expression compared with
control cells and a significant upregulation of HOXA7 (3.5-fold),
HOXA9 (2.0-fold), and MEIS1 (2.5-fold) (Figure 6D). The latter
suggests that HOX genes might be targets of MLL3 in FLT3-ITD
AML. 56 Moreover, depletion of wild-type MLL3 with murine
MLL3 siRNA in murine 32D myeloid cells stably expressing
murine FLT3-ITD increased cell growth in liquid culture and
clonogenic growth in soft-gel culture (Figure 6E-G) compared with
scramble siRNA-treated cells. In addition, FLT3-ITD patients with
MLL3 mutations had a worse prognosis compared with FLT3-ITD
patients without MLL3 mutations, as reflected by overall survival
and REL-free survival (Figure 6H-I). Together, these results strongly
suggest that MLL3 acts as a tumor suppressor gene that is frequently lost
in FLT3-ITD-mutant cells.
FAT mutations and XPO1 inhibition in FLT3-ITD AML
The FAT1 gene is located on human chromosome 4q35.2, a prevalent
region of deletion in human cancer.57-59 We identified and verified an
unexpectedly high rate (10%) of FAT1 mutations in FLT3-ITD samples
(8 of 80) (Figure 7A). TCGA data analysis showed focal deletion on
chromosome 4q35.2 containing FAT1 in 2% of AML samples (4 of
200) (Figure 7B) and a single patient with a missense mutation
(R1099P) (Figure 7A). Our SNP chip data also showed that 1 of 11
patients had a deletion of FAT1 at REL (supplemental Figure 14). To
study the biological role of FAT1 inactivation in FLT3-ITD, the expression of the FAT1 gene was silenced in MV4-11 cells by using
siRNA against FAT1 (Figure 7C). A significant increase in cellular
proliferation in liquid culture and soft-gel culture was observed in these
FAT1-depleted cells (Figure 7D-E).
TCGA data analysis showed that 3% of AML samples (6 of 200)
had focal deletion of FAT4 (supplemental Figure 15A). FAT4 was
moderately frequently mutated (5%; 4 of 80) in our FLT3-ITD samples,
occurring in a mutually exclusive manner to samples with FAT1 mutations (supplemental Figure 15B).
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Figure 5. MLL3 gene is mutated in FLT3-ITD AML both at DX and REL, and silencing of MLL3 in FLT3-ITD cells increased their growth in both liquid culture and
clonogenic assay. (A) Schematic of the MLL3 domains and locations of the amino acid substitutions caused by somatic mutations detected by WES and TDS. Black or red
triangles indicate missense mutations or nonsense mutations (either frameshift [fs] or stop-gain [X], respectively). Red arrow represents nonsense mutation identified in AML
TCGA data. Structural motifs of gene: A.T hook (ATPase a/b signature), PHD (plant homeodomain), DHHC (palmitoyltransferase activity), FYR (phenylalanine tyrosine-rich
domain), SET (suppressor of variegation, enhancer of zeste, trithorax). (B) Real-time PCR analysis showed reduced MLL3 mRNA in MLL3 shRNA-treated cells compared
with scramble shRNA-treated cells. MLL3 shRNA3 and MLL3 shRNA4 showed approximately 50% to 60% knockdown in MV4-11 cells compared with scramble shRNA. (C)
Western blot shows reduced MLL3 protein levels in MLL3 shRNA transduced cells (MV4-11) compared with scramble shRNA-treated cells. GAPDH is used as an internal
control. (D) Short-term cell proliferation assays of MV4-11 cells transduced with either MLL3 shRNAs or scramble shRNA. Data represent means 6 standard deviation (SD);
n 5 4. (E) For cell counting assay, 0.5 3 105 cells were plated in 6-well plates in quadruplets. Cell proliferation was measured by counting cells over a 5-day period. Results
are shown as means 6 SD; n 5 4. (F) Methylcellulose colony assay showed a significant increase in the number of MV4-11 colonies after cells were transfected with MLL3
shRNA compared with scramble shRNA-treated cells. Data represent means 6 SD; n 5 3. *P # .01; **P # .001.
Recent studies showed that XPO1 levels were higher in AML
patients with FLT3 mutations.60-62 Elevated levels of XPO1 in our
RNA sequencing data were confirmed by real-time data at REL compared with DX (supplemental Figure 16A). An XPO1 inhibitor
(KPT330) combined with standard AML induction chemotherapy is in
clinical trials. We found that KPT330 combined with standard AML
chemotherapy synergistically inhibited proliferation of FLT3-ITD
AML cells (supplemental Figure 16B-D).
Discussion
AML occurs because of a series of mutations and results in aberrant
proliferation and impaired differentiation of hematopoietic stem and
progenitor cells.63 Next-generation sequencing has allowed a highthroughput, comprehensive characterization of cancer genomes that
can aid in making clinical decisions. In the last few years, genes from
adult AML patients have been extensively sequenced.16-20 A few
reports included a limited number of DX and REL samples.23,64,65
However, none included DX and REL matched samples of FLT3-ITD
AML. In this study, we sequenced a very large FLT3-ITD cohort that
included DX and REL paired samples (supplemental Tables 1 and 6) to
discover the mutations associated with FLT3-ITD. We discovered that
the average number of coding mutations (single nucleotide variants and
indels) was 13.6 mutations per sample, which is comparable to AML in
general (;10 mutations per sample)23-27,30,34 and clearly lower than
that in solid tumors. All the mutations were validated by Sanger
sequencing. Recurrent mutations in 10 genes were also identified in the
REL-specific phase but not at DX in 50 trio (DX, CR, and REL)
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2498
GARG et al
BLOOD, 26 NOVEMBER 2015 x VOLUME 126, NUMBER 22
Figure 6. MLL3 acts as a recessive gene mutated in FLT3-ITD subgroup. (A) Xenografts (4 weeks) were established using MV4-11 cells stably expressing either scramble or
MLL3 shRNA. Scale is in centimeters. (B) Weight of individual tumors in each group (mean 6 SD; P 5 .0098). (C) Relative mRNA expression of MLL3 (quantitative PCR) in
xenografts. Values represent mean 6 SD; n 5 3. *P , .05. (D) Quantitative PCR showed relative increase in mRNA levels of HOXA7, HOXA9, and MEIS, and relative decrease in
the mRNA levels of p21 and p53 (growth-inhibitory genes). Data represent mean 6 SD; n 5 3. *P , .05; **P # .01. (E,F) Knockdown of murine MLL3 in 32D cells stably expressing
murine FLT3-ITD caused increased cell growth in liquid culture. Data represent mean 6 SD; n 5 3. *P # .05; **P # .01. (G) Methylcellulose colony formation assay of 32D cells
(stably expressing murine FLT3-ITD) transfected with either scramble siRNA or siRNA MLL3. Data represent mean 6 SD; n 5 3. *P # .05. (H) Survival curves of AML patients
either with or without MLL3 mutations. (I) Relapse-free survival curves of AML patients either with or without MLL3 mutations.
samples. This may be the result of small subclones having VAFs of less
than 5% at DX. Our sequencing depth (120X) limits the power to detect
subclones with VAFs of less than 5%. The previous reported high rate
of mutations of FLT3, NPM1, DNMT3A, IDH1, IDH2, TET2, ASXL1,
RUNX1, and WT1 genes highlights the power of WES/whole-genome
sequencing.23-27,66 However, the frequency of these mutations varied
remarkably between the genomes of normal karyotype FrenchAmerican-British M1 AML and M3 AML (PML-RARA fusion),67
suggesting that AML is heterogeneous and supports the further
characterization of different AML subtypes. In our study, mutations
were found in previously identified driver genes (NPM1, DNMT3A,
WT1, FLT3, TET2, IDH1, IDH2, RUNX1, ASXL1, and FAT4) as well as
either novel or previously unappreciated genes, including MLL3, FAT1,
SRCAP, NSD1, KDM6A, LRP2, and APOB (supplemental Table 16).
Several genes such as DNMT3A, NPM1, IDH1, IDH2, SF3B1, U2AF1,
and FLT3 (supplemental Figures 4 and 5) showed hotspot mutations,
mostly in line with earlier studies.
Comparative mutation analysis of primary and relapsed paired
samples showed high stability of mutations in DNMT3A, NPM1, IDH2,
RUNX1, and TET2, whereas less stability was observed in IDH1 and
FLT3-ITD (supplemental Figure 6). This was congruent with previously published studies.68-74
The overall mutational spectrum of 80 FLT3-ITD patients showed a
high prevalence of C.T transitions, but an increase in C.A and C.G
transversion occurred in REL compared with DX samples, suggesting
that chemotherapy has a potential effect on the mutational spectrum at
REL. Few copy-number changes were identified in REL of these
patients on the SNP chip, indicating that the increased rate of transversion was not associated with genomic instability. C.A transversions were frequent events in patients with AML and lung cancer (in
patients exposed to tobacco-borne carcinogens).23,75
The most represented signatures in our cohort were signature 1 and
signature 2. Signature 1 is associated with the age of the patients and is
dominant in various malignancies, including myeloid, breast, kidney,
and prostate cancer and glioblastoma.47,49 Signature 2 was enriched in
the REL samples, suggesting that this signature may be caused by
induction chemotherapy, but larger patient cohorts will be required to
confirm this observation.
Clonal expansion is a hallmark of cancers. Clonal evolution is
greatly influenced by chemotherapeutic agents because these agents are
continuously selecting resistance clones, the main basis for reoccurrence of disease.76 Our study extends the previous findings, which
recently described the pattern of clonal evolution in patients with acute
lymphocytic leukemia and AML by using fluorescence in situ
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BLOOD, 26 NOVEMBER 2015 x VOLUME 126, NUMBER 22
PROFILING SOMATIC MUTATIONS IN AML FLT3-ITD
2499
Figure 7. FAT1 and FAT4 somatic mutations in FLT3-ITD AML at DX and REL. (A) Schematic of FAT1 and FAT4 genes with the locations of alterations. Black or red
triangles show either missense or stop-gain, respectively. Red arrow represents missense mutation present in AML TCGA data set. Conserved domains are displayed by
using UniProt (http://www.uniprot.org/). (B) IGV (http://www.broadinstitute.org/igv) heat map of 4q35.2 shows deletional peak with FAT1 deletion culled from 200 AML patients
(TCGA consortium; http://cancergenome.nih.gov). (C) Real-time and protein blot display knockdown of FAT1 in MV4-11 cells. Quantitative PCR data represent means 6 SD;
n 5 3. **P # .01. (D) Cell growth showed increased proliferation with FAT1 knockdown in MV4-11 cells (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
assay; quadruplicate experiments). Data represent means 6 SD. *P # .05. (E) Cell counting assay: 0.5 3 105 cells were plated in 6-well plates in quadruplets, and cell
proliferation was measured by counting cells over a 5-day period. Results are shown as means 6 SD; n 5 4. (F) Methylcellulose colony assay of MV4-11 cells transfected with
either scramble control or FAT1 siRNA. Data represent means 6 SD; n 5 3. *P # .05.
hybridization, copy-number alterations (SNP arrays), and nextgeneration sequencing.23,77-80 We found that either the dominant clone
at DX gained additional mutations and expanded as an REL-specific
clone or a subclone at DX escaped chemotherapy and became the major
clone at REL. Taken together, these data suggest that relapsed AML
cells usually gain additional mutations during chemotherapy, possibly
contributing to drug resistance. In this context, 5 patients displayed
different ITD insertion sequences in the FLT3 gene between DX and
REL, suggesting that this is a possible mechanism of clonal evolution.
In our data, DNMT3Amut was found at a high VAF at DX and REL,
indicating that DNMT3Amut is part of the founder clone, and its
perseverance in CR revealed that the clone bearing DNMT3Amut may
survive chemotherapy and expand during CR. These findings suggest
that DNMT3Amut is one of the most durable and early mutations during
leukemogenesis.64,67,81-83 To achieve long-term disease-free survival,
future therapies need to eradicate all the preleukemic clones present at
DX that persist during treatment and contribute to REL.
Strikingly, mutations of different genes in the histone methyltransferase complex were mutually exclusive, suggesting that they have an
important role in pathogenesis of this disease. We observed recurrent
mutations as well as deletions in MLL3 in our FLT3-ITD cohort. MLL3
is one of the most frequently mutated and deleted genes in human
cancers including AML,84,85 but the biological significance of these
alterations is unknown. In our in vitro studies, selective inhibition of human
and murine MLL3 using siRNA in human MV4-11 cells (FLT3-ITD)
and murine cells (32D cells stably expressing murine FLT3-ITD)
accelerated their proliferation in liquid culture and colony formation in
methylcellulose gel. Moreover, stable knockdown of MLL3 in MV4-11
cells promoted tumor growth in a xenograft model. This suggests that
MLL3 acts as a tumor suppressor whereas loss in FLT3-ITD AML
contributes to the aggressive nature of the disease. More work is clearly
needed to test whether heterozygous FLT3WT/ITD knockin mice develop
leukemia when crossed with MLL3 heterozygous knockout mice.
Recent studies also suggest that MLL3 suppression alone is not
sufficient to drive leukemogenesis but instead cooperates with
p53 loss to block the differentiation of hematopoietic stem and
progenitor cells, leading to a myelodysplastic syndrome-like
disease.86 Another study showed that somatic cancer mutations of
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2500
BLOOD, 26 NOVEMBER 2015 x VOLUME 126, NUMBER 22
GARG et al
MLL3 resulted in loss of its enzymatic activity (H3K4 methylation), which affects the epigenetic landscape.87
FAT1 inhibition increased proliferation of MV4-11 cells in both
liquid and soft-gel culture. Mutual exclusivity between FAT1 and FAT4
suggests that either one of these genes enhances the progression of
leukemia. More research is required to test the biological role and
molecular mechanism of the FAT family in AML progression.
In summary, we reported the mutational landscape of the 80
FLT3-ITD cases and identified a number of novel driver genes. We also
highlighted a deregulated drug target that offers potential avenues for
treatment of AML, an aggressive leukemia. These data together provide
an enhanced road map for studying the molecular basis underlying this
deadly malignancy.
Acknowledgments
The authors thank Prof H. Serve and Dr J. Mueller for gifts of 32D
cells stably expressing murine wild-type FLT3 and FLT3-ITD, Ori
Kalid and Sharon Shacham for providing KPT330 (XPO1 inhibitor),
and Dr P. Tan for generously sharing related facilities.
This work was supported by National Institutes of Health (NIH)
National Cancer Institute grant R01CA026038-35 (H.P.K.), National Research Foundation Singapore and the Singapore Ministry
of Education under the Research Centres of Excellence initiative
(H.P.K.), the Singapore Ministry of Health’s National Medical
Research Council under its Singapore Translational Research
Investigator Award (H.P.K.), NSC91-2314-B-182-032 (Taiwan),
NHRI-EX96-9434SI (Taiwan), the Eleanor and Glenn Padnick
Discovery Fund in Cellular Therapy, and a generous donation from
the Melamed family.
Authorship
Contribution: M.G. and H.P.K. designed the study and wrote the
manuscript; M.G., D.K., Y.N., K.Y., Z.J.Z., S.H.K., L.-W.D., Q.-Y.S.,
D.-C.L. W.C., V.M., A.S., and S.V. performed the experiments; Y.N.,
A.M., K.Y., Y.O., Y.S., K.C., H.T., S.M., L.-Z.L., K.-T.T., M.S., and
H.Y. performed bioinformatic analysis; T.A., K.I., S.W., H.Y., W.J.C.,
S.-K.Y.K., A.E.-J.Y., J.S., K.-A.K., S.M.K., H.M.K., T.H., M.L.,
M.-C.K., L.-Y.S., I.-W.B., and O.B. coordinated sample collection and
processing; and M.G., Y.N., D.K., S.O., and H.P.K. analyzed and
discussed the data.
Conflict-of-interest disclosure: T.H. is employed by and partly
owns the MLL Munich Leukemia Laboratory. T.A. is employed by
the MLL Munich Leukemia Laboratory. The remaining authors
declare no competing financial interests.
Correspondence: Manoj Garg, Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational
Medicine, 14 Medical Drive MD-6, Singapore 117599; e-mail: csimg@
nus.edu.sg.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2015 126: 2491-2501
doi:10.1182/blood-2015-05-646240 originally published
online October 5, 2015
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Kantarjian, Torsten Haferlach, Michael Lill, Ming-Chung Kuo, Lee-Yung Shih, Igor-Wolfgang Blau,
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