1. Profiling of somatic mutations in acute myeloid leukemia with FLT3-ITD at diagnosis and relapse
by Manoj Garg, Yasunobu Nagata, Deepika Kanojia, Anand Mayakonda, Kenichi Yoshida, Sreya Haridas Keloth, Zhi Jiang Zang, Yusuke Okuno, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Ling-Wen Ding, Tamara Alpermann, Qiao-Yang Sun, De-Chen Lin, Wenwen Chien, Vikas Madan, Li-Zhen Liu, Kar-Tong Tan, Abhishek Sampath, Subhashree Venkatesan, Koiti Inokuchi, Satoshi Wakita, Hiroki Yamaguchi, Wee Joo Chng, Shirley-Kow Yin Kham, Allen Eng-Juh Yeoh, Masashi Sanada, Joanna Schiller, Karl-Anton Kreuzer, Steven M. Kornblau, Hagop M. Kantarjian, Torsten Haferlach, Michael Lill, Ming-Chung Kuo, Lee-Yung Shih, Igor-Wolfgang Blau, Olga Blau, Henry Yang, Seishi Ogawa, and H. Phillip Koeffler
Blood
Volume 126(22):
November 26, 2015
©2015 by American Society of Hematology

2. Somatic mutations identified by WES and TDS of FLT3-ITD AML
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).
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).
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

3. Distribution of somatic mutations in 80 patients with FLT3-ITD AML
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.
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.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

4. Distribution of mutational nucleotide classes between DX and REL paired samples.
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 5′ and 3′ 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.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

5. Clonal evolution from primary to REL in UPN001 and UPN002 and pattern of evolution observed in 13 DX and REL pairs.
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.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

6. 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.
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 α/β 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 ± standard deviation (SD); n = 4. (E) For cell counting assay, 0.5 × 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 ± SD; n = 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 ± SD; n = 3. *P ≤ .01; **P ≤ .001.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

7. MLL3 acts as a recessive gene mutated in FLT3-ITD subgroup.
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 ± SD; P = .0098). (C) Relative mRNA expression of MLL3 (quantitative PCR) in xenografts. Values represent mean ± SD; n = 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 ± SD; n = 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 ± SD; n = 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 ± SD; n = 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.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology

8. FAT1 and FAT4 somatic mutations in FLT3-ITD AML at DX and REL
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.
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 ( (B) IGV ( heat map of 4q35.2 shows deletional peak with FAT1 deletion culled from 200 AML patients (TCGA consortium; (C) Real-time and protein blot display knockdown of FAT1 in MV4-11 cells. Quantitative PCR data represent means ± SD; n = 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 ± SD. *P ≤ .05. (E) Cell counting assay: 0.5 × 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 ± SD; n = 4. (F) Methylcellulose colony assay of MV4-11 cells transfected with either scramble control or FAT1 siRNA. Data represent means ± SD; n = 3. *P ≤ .05.
Manoj Garg et al. Blood 2015;126:
©2015 by American Society of Hematology