Letters to the Editor 945 Whole-exome sequencing identifies a novel somatic in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia

Leukemia(2014) 28, 945–948; doi:10.1038/leu.2013.314 and no difference in quality was seen in the further analyses, including exome capture, PCR amplification and sequencing. To delineate the breakpoints on 1 and 22, 5 Acute myeloid leukemia (AML) is a clonal hematopoietic disease PCR was performed as described. Multiplex PCR was performed caused by both inherited and acquired genetic alterations. with combinations of primers (as shown in Figure 1d) to narrow Conventional cytogenetic analysis reveals chromosomal aberra- down the breakpoint region. When single primer pairs were tions in B50% of AML cases. These chromosomal rearrangements then used for individual PCR reactions, no amplification promote leukemia by affecting cell proliferation, differentiation occurred with OTT4 and In4.1–3, but 0.5 and 2.2 kb products and/or survival. In addition to these genetic alterations, cooperat- were amplified with OTT4 and In4.4 and In4.5, respectively ing are required for leukemogenesis.1–3 Among the (Figure 1e) confirming that the translocation occurred between subtypes of AML, pediatric-acute megakaryoblastic leukemia 1 and 22 with the breakpoint in intron 1 of OTT (AMKL) is primarily associated with three cytogenetic subtypes: and intron 4 of MKL1. one with trisomy 21 plus a mutation of GATA1, one with the To identify potential cooperating mutations in this patient t(1;22) translocation4,5 and a third recently reported recurrent sample, exome capture followed by deep sequencing was cryptic inversion of chromosome 16 (inv(16)(p13.3q24.3)).6,7 AMKL performed on the leukemia and remission blood samples from with t(1;22) is typically an aggressive disease, presenting in early the patient. A total of 88 807 512 and 79 716 978 reads were infancy with a median survival of just 8 months. Translocation obtained from the leukemic and remission samples, respectively between chromosomes 1p13 and 22q13 results in the fusion (Supplementary Table S2). Picard software identified and removed , RBM15-MKL1. The mechanism(s) by which the fusion PCR duplicates (14.0% of the leukemia sample and 9.4% of the promotes leukemia is unknown. RBM15-MKL1 knock-in mice blood sample). About 96% of the remaining unique reads from develop AMKL, but with delayed onset (16–18 months) and both samples mapped to only one location in the genome with 8 B incomplete penetrance. Induced RBM15-MKL1 overexpression in 94% of targeted bases with 410 Â coverage (Supplementary cell lines causes cell death.9 Together, these findings suggest that Table S2). Varscan identified 97 926 single nucleotide variations RBM15-MKL1 requires cooperating mutations to induce AMKL. To (SNVs) with a minimum variant frequency in the sample of 40.02 identify potential cooperating mutations in t(1;22) AMKL, we and minimum average quality score of 420. Of these variants, performed a whole-exome capture followed by next-generation 22 326 were called somatic variations, which were filtered to sequencing in diagnostic and remission samples from a t(1;22)- require o0.2 P-value so that the SNV was different in leukemia vs AMKL patient. An 11-week-old boy presented with loss of appetite remission, leaving 4580. Of the 4580 SNV, 2123 were in genic and fussiness. Samples of bone marrow and peripheral blood were regions and 95.3% were classified as coding variants, with 75.2% obtained at presentation. Morphological and immunophenotypic being non-synonymous. SIFT (sorting intolerant from tolerant) analyses were performed at the UC Davis Pathology Department predicted 36.0% and 62.9% of the coding variants as damaging according to standard protocols. FISH and cytogenetic analyses and tolerated variations, respectively (Supplementary Table 3). were performed by the Mayo Clinic Laboratory (Children’s Of 1956 coding variants, 1876 were novel SNPs based on dbSNP. Oncology Group designated laboratory). The remaining Of the 1956, 377 SNVs remained after further filtering to require diagnostic sample was frozen. The blood had a white blood cell o0.05 P-value and damaging/tolerated SIFT predictions, and to count of 38 000/mm3, hemoglobin of 6.1 g/dl and platelet count of exclude known dbSNPs. To overcome the low blast purity 99 000/mm3. The peripheral blood smear showed immature blast (15% of cells), we used a less stringent filter to maximize the cells (18% of white blood cells). Bone marrow examination chances of detecting variations within the leukemic cells, which revealed severe fibrosis (Figures 1a and b). Peripheral blood resulted in false positive variants from the initial analysis. immunophenotyping showed 15% leukemic blasts based on Therefore, it was necessary to carefully inspect the reads CD45 expression and Side Scatter profile. These cells expressed manually using the integrated genomics viewer. After manual CD33 (75%), CD34 (44%), CD41 (63%) and CD61 (79%), and inspection of the 377 SNVs for base quality displayed in the cytogenetic analyses showed 46,XY,t(1;22)(p13;q13) (Figure 1c). integrated genomics viewer (IGV v2.1,10), we found 12 variants Based on the fibrotic marrow, megakaryocytic markers and to be likely candidates and performed further validation for translocation, the diagnosis of AMKL was assigned. He responded these 12 variants (Table 1). Neither known AML-associated very well to the treatment with cytarabine, etoposide, daunorubicin, mutations nor mutations in tumor suppressor were found. mitoxantrone, L-asparaginase and Gemtuzumab, defined in the However, small indels would not be detectable in our data Children’s Oncology Group AAML0531 protocol. Because he did not owing to the low blast percentage. have an HLA matched family donor, he did not undergo allogeneic To validate the candidate mutations, Sanger sequencing was hematopoietic stem cell transplantation. He is currently disease free performed, but proved to be technically challenging owing to the 4 years after completing therapy. Peripheral blood was collected and low percentage of blasts. By Sanger sequencing, the expected cryopreserved 6 and 9 months after remission was achieved. peak for the variant alleles was not distinguishable except for The quality of extracted genomic DNA from the fixed cells one in HERC2 that proved to exist in both the leukemic and ofthediagnosticsampleusingtheGentraPuregenkit remission samples at B50%, suggesting a unique SNP in the (Qiagen, Germantown, MD, USA) was highly comparable to that patient (data not shown). To improve the sensitivity, 10 of the of freshly thawed peripheral blood of the remission sample remaining 11 variants were further validated with droplet digital (A260/280 ¼ 1.89 vs 1.86, leukemia and remission, respectively) PCR (one failed in the assay design because of repetitive

Accepted article preview online 25 October 2013; advance online publication, 22 November 2013

& 2014 Macmillan Publishers Limited Leukemia (2014) 935 – 979 Letters to the Editor 946

Figure 1. Analysis of t(1;22) sample and functional analysis of MMP8G189D.(a, b) Blasts from a touch-prepped bone marrow (original magnification  100) and severe fibrosis in bone marrow core (original magnifications  50), respectively. (c) G-banded karyotype with t(1;22)(p13;q13) at diagnosis from the patient. Arrows indicate the rearranged chromosomes. (d) Diagram of primer bound locations on the OTT gene and MKL1 gene. Red lightening symbols indicate the breakpoint. (e) PCR amplicon of OTT4/IN4.3 was not observed in lane 1 but PCR amplicons of OTT4/IN4.4 and OTT4/IN4.5 were observed at around 500 bp and 2.2 kb, in lane 2 and 3, respectively. This analysis indicated that the breakpoint is between IN4.3 and IN4.4 in MKL1 intron 4. (f) Western blot was probed using an anti-flag antibody on the whole-cell lysates and immunoprecipitates by flag beads of HEK 293T cells carrying the empty vector (empty), wild-type (wt) and mutant (G189D) MMP8. (g) Type 1 collagen substrate zymography showed the reduced enzymatic activity of mutant MMP8 (G189D) compared with wild-type MMP8 (wt). (h) The degraded areas by MMP8 were calculated using ImageJ.

Table 1. Somatic mutations identified by exome sequencing and ddPCR

Position Gene Ref/ Substitution P-value No. of Mut SIFT Allele frequency symbol mut reads prediction Exome sequencing ddPCR

Leukemic Remission Leukemic Remission

chr11:102592188 MMP8 C/T G189D 2.47E À 06 18 Damaging 14.40% 0.00% 14.50% 0% chr15:75684939 SIN3A G/T S832* 4.33E À 02 5 Nonsense 3.47% 0.00% 0% 0% chr18:52899759 TCF4 T/G I544L 4.60E À 03 9 Tolerated 4.46% 0.00% 0% 0% chr5:140230206 PCDHA9 G/T S709I 2.87E À 02 4 Damaging 4.76% 0.00% 0% 0% chr1:38022574 DNALI1 G/T W15C 4.50E À 02 4 Tolerated 4.35% 0.00% 0% 0% chr11:60638721 ZP1 G/T W349L 4.06E À 02 4 Damaging 4.60% 0.00% 0% 0% chr19:48305564 TPRX1 T/A N235I 1.79E À 03 8 Tolerated 7.69% 0.00% N/A N/A chr15:28456220 HERC2 G/A L2333F 5.05E À 02 5 Tolerated 4.50% 0.00% 50% 50% chr20:44579096 ZNF335 G/T Q887K 5.37E À 02 3 Tolerated 3.45% 0.00% 0% 0% chr3:146246571 PLSCR1 G/T P48T 4.47E À 02 4 Tolerated 7.14% 0.00% 0% 0% chr3:42730118 KBTBD5 G/T E444* 5.30E À 02 3 Nonsense 4.55% 0.00% 0% 0% chr9:130550569 CDK9 T/C L170P 5.23E À 02 4 Damaging 7.84% 0.00% 0% 0% Abbreviations: ddPCR, droplet digital PCR; N/A, not applicable; SIFT, sorting intolerant from tolerant.

sequences and a high GC content). Despite multiple repetitions CDK9 using exome sequencing, we had hypothesized that this and assessment using modified primer pairs, of the 10 variants was a heterozygous mutation in the leukemic blasts. However, this assessed using droplet digital PCR, only MMP8 was validated was not validated with droplet digital PCR even after analysis of (Supplementary Figure S1), whereas the other nine variants were over 14 000 molecules of this region from the diagnostic DNA not detectable in the leukemic sample (Table 1). Given that, for sample (Supplementary Figure S1). Although CDK9 is known to example, a candidate mutation with 7% frequency was found in have a role in megakaryocyte maturation and to regulate both the

Leukemia (2014) 935 – 979 & 2014 Macmillan Publishers Limited Letters to the Editor 947 cell cycle and transcription, further inspection of the exome data by the Connecticut Stem Cell grant no. 10SCB03 (DSK), NIH R01HL106184 (PGG), showed that the sequence quality was low at the mutant bases. a National Research Service Award from NIH/NCI F32CA165683 (YK), Hyundai Other unvalidated variant calls had low number of mutant reads, Hope on Wheels Award (NS and TAG), the Eric Trump Foundation (TAG) and low mutant base quality or possible mapping problems. The theYaleStemCellCenterCorefundedbytheConnecticutStemCellResearch droplet digital PCR validation demonstrated that a more stringent Fund.

P-value, mutant base quality and read number filtration is 1,2 3 4 5,6 1,2,7 necessary when analyzing challenging low-blast percentage Y Kim , VP Schulz , N Satake , TA Gruber , AM Teixeira , S Halene8, PG Gallagher3,9 and DS Krause1,2,10 samples. 1 The MMP8 mutation in our patient sample, a substitution Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA; from C to T on chr11:102592188, resulted in an amino-acid 2 change MMP8G189D. This mutation is predicted to be damaging Yale Stem Cell Center, Yale University School of Medicine, 11,12 New Haven, CT, USA; and deleterious by SIFT and PROVEAN, respectively. 3 This region is universally conserved in all vertebrates except Department of Pediatrics, Yale University School of Medicine, 13 New Haven, CT, USA; in elephants and frogs (http://genome.uscs.edu/, ). MMP8 4 encodes matrix metalloproteinase-8, which breaks down type Section of Hematology/Oncology, Department of Pediatrics, I, II and III collagens in the extracellular matrix, and requires both University of California, Davis, Comprehensive Cancer Center, 14 Sacramento, CA, USA; calcium and zinc ions for activity. The variation is located at a 5 calcium ion binding site in the catalytic domain (Supplementary Departments of Oncology, St Jude Children’s Research Hospital, Memphis, TN, USA; Figure S2). To determine the effect of this mutation on 6 enzymatic activity, we generated constructs containing either Department of Pathology, St Jude Children’s Research Hospital, Memphis, TN, USA; wild-type or mutant MMP8 and expressed them in HEK 293T 7 cells. No difference was observed in the expression levels Department of Pathology, Yale University School of Medicine, New Haven, CT, USA; between the constructs (Figure 1f). Type 1 collagen substrate gel 8 zymography revealed significantly reduced proteolytic activity Yale Comprehensive Cancer Center, Division of Hematology, of the mutant compared with wild-type MMP8 (Figures 1g and Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA; h, P-value ¼ 0.017). To further assess whether MMP8 may have a 9 role in AMKL leukemogenesis, we evaluated MMP8 expression in Department of Genetics, Yale University School of Medicine, New Haven, CT, USA and primary AMKL samples. Although RNA was not available from 10 the index case, gene expression data from 41 pediatric Department of Cell Biology, Yale University School of Medicine, AML cases including 16 cases of AMKL demonstrated eight of New Haven, CT, USA the 16 AMKL cases to have detectable MMP8 expression E-mail: [email protected] (Supplementary Figure S3).7 All AMKL samples in this study had 490% purity, an important consideration given that MMP8 is known to be highly expressed in mature myeloid cells. Transcriptome sequencing was available for 14 of these AMKL cases and the sample with the highest signal by exon array also REFERENCES had significant levels of the MMP8 transcript by sequencing. In 1 Ding L, Ley TJ, Larson DE, Miller CA, Koboldt DC, Welch JS et al. Clonal evolution in an independent cohort of 60 AMKL cases also analyzed by relapsed acute myeloid leukaemia revealed by whole-genome sequencing. gene-expression profiling, a subset of patients had evidence of Nature 2012; 481: 506–510. 15 MMP8 expression. Although the purity of these samples was 2 Fro¨hling S, Scholl C, Levine RL, Loriaux M, Boggon TJ, Bernard OA et al. extremely variable (12–94%), several samples with low purity Identification of driver and passenger mutations of flt3 by high-throughput dna (M7122, 12%; M7026, 28%) had low levels of MMP8 expression sequence analysis and functional assessment of candidate alleles. Cancer Cell demonstrating that the variability in MMP8 expression cannot 2007; 12: 501–513. solely be due to myeloid cell contamination. Furthermore, there 3 Walter MJ, Ding L, Shen D, Shao J, Grillot M, McLellan M et al. Recurrent DNMT3A are cases with elevated expression that are relatively pure mutations in patients with myelodysplastic syndromes. Leukemia 2011; 25: 1153–1158. (M7040, 70%; M7072, 90%). Although the same mutation was 4 Baruchel A, Daniel MT, Schaison G, Berger R. Nonrandom t(1;22)(p12-p13;q13) in not found in any of the samples, MMP8 is aberrantly expressed acute megakaryocytic malignant proliferation. Cancer Genet Cytogenet 1991; 54: in a subset of AMKL cases. In addition, MMP8 mRNA is expressed 239–243. in murine megakaryocyte-erythroid progenitors based on the 5 Mercher T, Busson-Le Coniat M, Khac FN, Ballerini P, MauchauffE` M, Bui H et al. ENCODE database (GSE40522). Given that MMP8 was the only Recurrence of OTT-MAL fusion in t(1;22) of infant AML-M7. Genes Chromosomes candidate gene with 14% variation frequency in the exome Cancer 2002; 33: 22–28. sequencing, the patient had few cooperating mutations in 6 Thiollier C, Lopez CK, Gerby B, Ignacimouttou C, Poglio S, Duffourd Y et al. coding regions, which might explain the excellent response to Characterization of novel genomic alterations and therapeutic approaches treatment and survival. In conclusion, we provide the first data using acute megakaryoblastic leukemia xenograft models. J Exp Med 2012; 209: 2017–2031. identifying a novel somatic mutation that may provide a key 7 Gruber Tanja A, Larson Gedman A, Zhang J, Koss Cary S, Marada S, Ta Huy Q et al. insight to understanding the pathogenesis of RBM15-MKL1 in An Inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an AMKL. aggressive subtype of pediatric acute megakaryoblastic leukemia. Cancer Cell 2012; 22: 683–697. 8 Mercher T. The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model. J Clin CONFLICT OF INTEREST Invest 119: 852–864. The authors declare no conflict of interest. 9 Descot A, Rex-Haffner M, Courtois G, Bluteau D, Menssen A, Mercher T et al. OTT-MAL is a deregulated activator of -dependent gene expression. Mol Cell Biol 2008; 28: 6171–6181. 10 Thorvaldsdo´ttir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform ACKNOWLEDGEMENTS 2013; 14: 178–192. We thank Dr Yardena Samuels (Weizmann Institute of Science, Rehovot, Israel) 11 Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of for providing protocols for collagen zymography. This work was supported amino acid substitutions and indels. PLoS One 2012; 7: e46688.

& 2014 Macmillan Publishers Limited Leukemia (2014) 935 – 979 Letters to the Editor 948 12 Choi Y. A fast computation of pairwise sequence alignment scores between a 14 Massova I, Kotra LP, Mobashery S. Structural insight into the binding motifs for protein and a set of single-locus variants of another protein. In: Proceedings of the the calcium ion and the non-catalytic zinc in matrix metalloproteases. Bioorg Med ACM Conference on Bioinformatics, Computational Biology and Biomedicine. ACM: Chem Lett 1998; 8: 853–858. Orlando, FL, USA, 2012; pp 414–417. 15 Bourquin JP, Subramanian A, Langebrake C, Reinhardt D, Bernard O, Ballerini P et al. 13 Dreszer TR, Karolchik D, Zweig AS, Hinrichs AS, Raney BJ, Kuhn RM et al. The UCSC Identification of distinct molecular phenotypes in acute megakaryoblastic Genome Browser database: extensions and updates 2011. Nucleic Acids Res 2012; leukemia by gene expression profiling. Proc Natl Acad Sci USA 2006; 103: 40: D918–D923. 3339–3344.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Synergistic anti-leukemic activity of imatinib in combination with a small molecule Grb2 SH2 domain binding antagonist

Leukemia (2014) 28, 948–951; doi:10.1038/leu.2013.323 combination of these two agents increased apoptosis by 450% (20% vs 36%; Figure 1d), indicating functional complementation of distinct pro-apoptotic effects. Flow cytometry analysis using CellTrace Violet indicated that vehicle-treated K562 cells divided eight times in 72 h. Treatment with 0.25 or 2.5 mM imatinib alone The mechanisms underlying imatinib resistance in chronic resulted in 0.04% and 0.03% of cells reaching the eighth cell myelogenous leukemia (CML) include BCR-ABL gene rearrange- division, respectively, and treatment with 2 mM TB03 alone resulted ment, overexpression of Bcl-Abl tyrosine kinase fusion protein and in 0.08% of cells reaching the eighth cell division (Figure 1e). activation of Bcr-Abl-independent pathways such as those of Src Combined imatinib/TB03 treatments further reduced the number kinases.1,2 Although impressive therapeutic responses have been of cells reaching eight divisions: 0.02% for 2 mM TB03 plus 0.25 mM achieved with imatinib treatment, many patients fail to respond imatinib and 0.01% for 2 mM TB03 plus 2.5 mM imatinib; the latter optimally due to innate or acquired resistance.1,2 The available combination represents an eightfold reduction in cell survival treatments for these patients are limited and improved treatment (Figure 1e). Short-term analysis of cell cycle progression using options are urgently needed. In patients with Philadelphia Vybrant DyeCycle Green showed that imatinib treatment chromosome-positive CML, tyrosyl phosphorylation of the increased the G0/G1 population while decreasing the S and Bcr-Abl oncoprotein enables binding to the Src homology-2 (SH2) G2/M phase populations, consistent with cell cycle suppression domain of the adapter protein growth factor receptor-bound 2 (Figure 1f). Treatment with TB03 alone had little effect, but (Grb2), thereby activating downstream signaling pathways, combined TB03/imatinib treatment increased the number of cells including the Ras/MAPK pathway, controlling cell proliferation.3 in G0/G1 in a dose-dependent manner, with proportionately fewer Grb2 mutants missing one of its two SH3 domains impair cell cells reaching S and G2/M. The increased number of cells in transformation by Bcr-Abl, highlighting the importance of Grb2 sub-G1 phase with imatinib treatment relative to control (1.5% vs interactors, such as SOS1, in oncogenic signaling.4 These findings 4.7%) may reflect pro-apoptotic effects. Consistent with the also suggest that Grb2 binding antagonists may provide an complementation of apoptosis observed by annexin staining, effective alternative or adjunct therapeutic strategy for CML TB03 plus imatinib further increased the number of cells in this patients resistant to imatinib. phase in a dose-dependent manner, from 4.7 to 7% for Small molecule Grb2 SH2 domain binding antagonists have imatinib:TB03 at a ratio 0.5:6.25 mM and from 4.7 to 11% for been developed that are selective, potent and phosphatase imatinib:TB03 at 0.5:12.5 mM (Figure 1f). These results reinforce the resistant.5 These have been shown to block the growth of tumor- hypothesis that TB03 alone inhibits only cell cycle progression, but derived cells in culture as well as tumor angiogenesis and enhances imatinib-induced apoptosis and cell cycle suppression, metastasis in mice.3,5,6 We report here that TB03 (Supplementary resulting in synergistic inhibition of K562 proliferation. Figure 1), a member of this compound class, synergistically Cancer stem cells (CSCs) have become prime suspects for enhanced inhibition of K562 leukemia cell proliferation by mediating resistance to therapy, disease relapse and progression.7 imatinib (Figure 1). Treatment with TB03 or imatinib alone High levels of aldehyde dehydrogenase (ALDH) activity have been resulted in dose-dependent growth inhibition (Figures 1a and recognized as a CSC marker in many cancers, including leukemia.8 b); when combined with TB03, imatinib activity was significantly The effects of imatinib, TB03 and combined imatinib/TB03 enhanced (Figure 1b). Combining imatinib with TB03 at a ratio of treatment on a putative leukemia stem cell subpopulation of 1:100 was synergistic and achieved an ED90 with a combination K562 was investigated by flow cytometry using ALDEFLUOR, index (CI) of 0.774. At ratios of 1:50, 1:25, 1:12.5 and 1:6.25, all which provides a fluorescent ALDH reaction product to identify combinations were synergistic as indicated by CIs at ED50,ED75 or stem and progenitor cells by ALDH activity. Among vehicle- þ ED90 values (Supplementary Table 1 and Figure 1c). treated K562 cells 11.4% were ALDH , this was unaltered by TB03 Flow cytometry experiments were performed to further treatment but decreased to 8.9% and 2.7% with imatinib characterize synergistic inhibition of K562 cell proliferation by treatment at 0.25 and 25 mM, respectively (Supplementary the combination of imatinib and TB03. Analysis using annexin Figure 2). When combined with TB03 (20 mM), imatinib at 0.25 þ V–fluorescein isothiocyanate/propidium iodide double staining and 25 mM concentrations further reduced the ALDH K562 showed that treatment with either imatinib or TB03 alone did not population to 5.0% and 1.8%, respectively (Supplementary affect apoptosis relative to the vehicle control group, but the Figure 2). To our knowledge, this is the first evidence of leukemia

Accepted article preview online 31 October 2013; advance online publication, 22 November 2013

Leukemia (2014) 935 – 979 & 2014 Macmillan Publishers Limited