Summary The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSC). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSC. Inactivation of Dot1l lead to down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global gene expression remained largely unaffected. Suppression of MLL-translocation associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

Summary We created a mouse model where conditional expression of an Mll-AF4 fusion oncogene induces B-precursor acute lymphoblastic (ALL) or acute myeloid leukemias (AML). Gene expression profile analysis of the ALL cells demonstrated significant overlap with human MLL-rearranged ALL. ChIP-chip analysis demonstrated histone H3 Lysine 79 (H3K79) methylation profiles that correlated with Mll-AF4 associated gene expression profiles in murine ALLs, and in human MLL-rearranged leukemias. Human MLL-rearranged ALLs could be distinguished from other ALLs by their H3K79 profiles and suppression of the H3K79 methyltransferase DOT1L inhibited expression of critical MLL-AF4 target genes. We have thus demonstrated that ectopic H3K79 methylation is a distinguishing feature of murine and human MLL-AF4 ALLs and is important for maintenance of MLL-AF4 driven gene expression. Significance The t(4;11) encodes an MLL-AF4 fusion protein, and predicts a particularly poor prognosis when found in acute lymphoblastic leukemias (ALL). Recent studies suggest certain MLL-fusion proteins enhance gene expression by recruitment of the histone H3 lysine79 (H3K79) methyltransferase DOT1L. We demonstrate that H3K79 methylation is enhanced at many loci in leukemia cells from a murine model of Mll-AF4 and in human MLL-AF4 leukemia cells and this elevation is correlated with enhanced gene expression. Furthermore, suppression of H3K79 methylation leads to inhibition of gene expression in MLL-AF4 cells. These data demonstrate that inhibition of DOT1L may be a therapeutic approach in this disease, and that this mouse model should be useful for assessment of therapeutic approaches for MLL-rearranged ALL.

IntroductionFMS-like tyrosine kinase-3 (FLT3) has been shown to be mutated in about one-third of patients with acute myeloid leukemia (AML), representing one of the most frequently occurring mutations in this disease. 1,2 Until now, two distinct clusters of activating mutations are known: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and point mutations (PMs) in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients. [3][4][5][6][7][8][9] Recently, the crystal structure of the autoinhibited form of FLT3 was resolved. 10 The structure conforms to the prototypical conformation common to other inactive kinases that have a "closed" activation loop, but the remarkable feature is the complete JM domain serving as a critical autoinhibitory loop and interacting with all key features of FLT3. This domain can be divided into three distinct parts: the JM binding motif (JM-B), JM switch motif (JM-S), and the zipper or linker peptide segment (JM-Z). According to that model, the JM-B region is nearly buried in the FLT3 structure. It serves as an autoinhibitory domain, which in an inactive state prevents the N lobe from rotating toward the C lobe of the tyrosine kinase domain (TKD) to generate the activated kinase fold.The cytoplasmatic juxtamembrane domain is highly conserved between different members of the class III receptor tyrosine kinase (RTK) family. A variety of tumors in animals and humans have been described that harbor activating mutations in the JM domain. [11][12][13][14] The most frequently occurring activating mutations in AML, FLT3-ITDs, occur primarily in the JM-Z domain. They represent a heterogenous group of mutations, where a fragment of the JM domain, varying in length from 2 to 204 nucleotides (nt), is duplicated and inserted in a direct head-to-tail orientation always maintaining the reading frame.Recently, we discovered a novel missense point mutation in the JM domain of FLT3 in the AML cell lines Mono-Mac (MM)-1 and MM-6, changing valine with alanine at position 592. 15 By performing a LightCycler (Roche, Mannheim, Germany) mutational screening of FLT3 in 785 AML patient samples, we were able to identify two other point mutations: F594L in two AML patients and Y591C in 1 AML patient. In addition, Stirewalt et al 16 found additional point mutations in the JM domain of FLT3 (V579A and F590GY591D) in AML patients by using single-stranded conformational polymorphism analyses (polymerase chain reaction [PCR]/SSCPs).Here, we have studied the functional significance of this new class of activating mutations in patients with AML: PMs that cluster in a 16-aa stretch of the JM domain (FLT3-JM-PMs).We could clearly demonstrate that FLT3 receptors harboring one of these JM point mutations, when expressed in Ba/F3 cells, Supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Sp556/3-1) and the Deutsche Krebshilfe (10-1997-Sp2).Reprints: Karsten Spiekermann, Department of Medicine III, University Hospital Grosshadern, CCG "Leukemia," GSF...

Purpose: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. Experimental Design: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. Results: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMSlike tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sitesY589 and Y599 and colocalized in vivo. Expression of CBLDexon8 and CBLDexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor^independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand^dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). Conclusion: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.CBL, a known negative regulator of activated receptor tyrosine kinases (RTK), is localized on human chromosome 11q23, a region frequently associated with chromosomal aberrations. Translocations t(4;11) and t(11;14), and mixed-lineage leukemia fusion genes involving CBL have been described in patients with leukemia and lymphoma (1 -3). CBL oncogenes were initially identified in the murine system. CBL-70Z, carrying an internal deletion of 17 amino acids, was isolated from the 70Z/ 3 mouse pre-B-cell lymphoma cell line (4). CBL-70Z deregulates the cellular tyrosine kinase machinery, as NIH3T3 serumstarved cells expressing CBL-70Z showed significantly increased endothelial growth factor receptor (EGFR) kinase activity after EGF stimulation (5). p95CBL, expressed in the murine reticulum sarcoma cell line J-774, lacks internal 111 amino acids, comprising whole exons 8 and 9 (6). CBL70Z and p95CBL mutations both target the linker and RING finger domain, which points to a mutation-sensitive region within the CBL protein. Recently the first human CBL mutation has been reported in a patient...

Purpose Histone deacetylase inhibitors (HDACi) have recently emerged as efficacious therapies that target epigenetic mechanisms in hematologic malignancies. One such hematologic malignancy, B-cell acute lymphoblastic leukemia (B-ALL), may be highly dependent on epigenetic regulation for leukemia development and maintenance, and thus sensitive to small molecule inhibitors that target epigenetic mechanisms. Experimental Design A panel of B-ALL cell lines was tested for sensitivity to HDACi with varying isoform sensitivity. Isoform specific shRNAs were used as further validation of HDACs as relevant therapeutic targets in B-ALL. Mouse xenografts of B cell malignancy derived cell lines and a pediatric B-ALL were used to demonstrate pharmacological efficacy. Results Non-selective HDAC inhibitors were cytotoxic to a panel of B-ALL cell lines as well as to xenografted human leukemia patient samples. Assessment of isoform specific HDACi indicated that targeting HDAC1-3 with class I HDAC specific inhibitors was sufficient to inhibit growth of B-ALL cell lines. Furthermore, shRNA mediated knockdown of HDAC1 or HDAC2 resulted in growth inhibition in these cells. We then assessed a compound that specifically inhibits only HDAC1 and HDAC2. This compound suppressed growth and induced apoptosis in B-ALL cell lines in vitro and in vivo while it was far less effective against other B-cell derived malignancies. Conclusions Here we show that HDAC inhibitors are a potential therapeutic option for B-ALL, and that a more specific inhibitor of HDAC1 and HDAC2 could be therapeutically useful for patients with B-ALL.

Purpose: Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT. Experimental Design: We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Y tyrosine kinase domain (TKD) mutant. All tyrosine residues in the juxtamembrane domain (Y566, Y572, Y589, Y591, Y597, and Y599), interkinase domain (Y726 andY768), and COOH-terminal domain (Y955 andY969) of the FLT3-D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplication mutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor. Result: The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role of Y589 and Y591in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WT and the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation. Conclusion: We identified two tyrosine residues,Y589 and Y591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WTand the transforming potential of oncogenic FLT3 mutants.FLT3 is a member of the class III protein receptor tyrosine kinase family (RTK) that is characterized by five extracellular immunoglobulin-like domains, a juxtamembrane domain (JM), and two protein tyrosine kinase domains (TKD) split by an interkinase domain (IK; ref. 1). The class III receptors also include KIT, FMS, platelet-derived growth factor receptor-a (PDGFRA), and platelet-derived growth factor receptor-h (PDGFRB). Binding of FLT3 ligand (FL) to its receptor induces dimerization, phosphorylation, and subsequent activation of downstream signaling pathways such as signal tranducer and activator of transcription 5 (STAT5), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/AKT (2 -6). FLT3 has been shown to play an important role in normal hematopoiesis and is highly expressed in CD34 + hematopoietic progenitor cells (2, 7 -9).Activating mutations of FLT3 are found in 30% of patients with acute myelogenous leukemia (AML) and are associated with an inferior clinical outcome (10 -12). FLT3 internal tandem dup...

FLT3-internal tandem duplications (FLT3-ITDs IntroductionMutations in the FMS-like tyrosine-kinase 3 (FLT3) are one of the most frequently found genetic alterations in patients with acute myeloid leukemia (AML), 1-13 myelodysplastic syndromes (MDSs; 10%-15%), 2,14 and acute lymphoblastic leukemia (ALL; 1%-3%). 9,16,17 FLT3 belongs to the class III of receptor tyrosine kinases, which are characterized by the presence of an extracellular immunoglobulin-like domain, a transmembrane and the cytoplasmic juxtamembrane (JM) domain, and the tyrosine kinase domain (TKD). 18 The class III receptors also include KIT, CSF-1, PDGFRA, and PDGFRB. 19,20 Activation of FLT3 by FLT3 ligand (FL) leads to receptor oligomerization and transphosphorylation of specific tyrosine residues, 21 which activates the downstream signaling pathways including STAT5, Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/AKT. [22][23][24][25] FLT3 is highly expressed in CD34 ϩ hematopoietic progenitor cells and plays an important role in normal hematopoiesis. [26][27][28][29] Three distinct activating mutations of FLT3 in hematologic malignancies have been reported: point mutations (FLT3-JM-PM) 30,31 and internal tandem duplications (FLT3-ITD) in the JM domain and mutations in the tyrosine-kinase domain (FLT3-TKD). 1,8,9,12,16,17,32 The crystal structure of FLT3 has shown that the JM domain acts as an autoinhibitory domain in the inactive state. 33 The JM domain is highly conserved across all members of class III RTKs. Hence many tumors in humans show activating mutations of JM in class III RTKs. 34-37 FLT3-ITDs, found in a majority of acute leukemia patients, are in-frame duplications of a fragment of the JM domain. FLT3-ITDs are highly heterogeneous and vary in length from 2 to 68 AAs. These duplications are thought to disrupt the autoinhibitory mechanism and result in constitutive activation of the catalytic domain of FLT3. Activated FLT3 mutants promote cell proliferation and inhibit apoptosis, leading to factorindependent growth of murine hematopoietic cells in vitro and a myeloproliferative phenotype in vivo. 38 FLT3-ITDs are present in the leukemic blasts of 20% to 30% of all AML patients. Recent studies have also shown that FLT3-ITDs are found in the leukemic stem cells. 39 Furthermore, the presence of a FLT3-ITD has been recognized as an independent poor prognostic factor in AML and is associated with a decreased survival due to an increased relapse rate. 8,9,11,12,[40][41][42][43] Several factors influence the poor prognosis seen in AML patients harboring FLT3-ITDs (eg, a high FLT3-ITD/wildtype ratio). 9,44 A recent study has reported that the detection of FLT3-ITD mutation in less mature progenitor populations, for example, CD34 ϩ /CD33 Ϫ , might be associated with drug resistance. 43 In the present study, we asked whether any common duplicated motif exists in AML patients carrying FLT3-ITDs, which might be responsible for the transforming potential. To address this question, we sequenced and analyzed th...

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