Mixed Lineage Leukemia Leukemia Research Articles

Bimodal Degradation of MLL Protein by the Cell Cycle SCFSkp2 and APCCdc20 E3 Lilgases Assures Cell Cycle Execution: A Critical Regulatory Circuit Lost in Leukemogenic MLL-Fusions.

The MLL (mixed lineage leukemia) gene encodes a highly conserved 3,969 aa histone H3 K4 methyl transferase, of which chromosome translocations result in poor prognostic infant and therapy-related leukemias. Mysteriously, the pathognomonic MLL leukemia fusions consist of a common amino(N)-terminal ∼1400 aa of MLL fused in frame with more than 60 different partners with no shared characteristics. The most well known genetic function of MLL is to antagonize polycomb group of proteins for proper Hox gene expression. Consequently deregulated Hox genes caused by MLL translocations contribute to the MLL leukemogenesis. Besides Hox genes, it remains largely undetermined whether MLL participates in other biological processes. The 500kD MLL precursor undergoes evolutionarily conserved proteolytic maturation mediated by Taspase1 (Hsieh et al. 2003, MCB, 23, 186–194; Hsieh et al. 2003, Cell, 115, 293–303). Our recent studies on Taspase1 knockout cells established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins and possibly Cdk inhibitors (Takeda et al. 2006, Genes & Development , 20, 2397–2409). As MLL actively participates in the cell cycle regulation, we investigated the regulation of MLL through cell cycle transition. We uncovered a unique biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M phase progression. Conversely, over-expression of MLL blocks S phase progression. Remarkably, MLL degradation initiates at its N-terminal ∼1400 aa that is retained in all MLL leukemia fusions. We examined prevalent MLL-fusions, including MLL-AF4, MLL-AF9, MLL-ELL and MLL-ELL, and observed their increased resistance to degradation. Furthermore, the same resistance was observed with the leukemogenic MLL-lacZ but not the non-leukemogenic MLL-Myc tag fusion. Thus, non-oscillating expression of MLL-fusions through the cell cycle, resulted from impaired degradation, likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) to assure the temporal necessity of MLL in coordinating cell cycle progression. Future studies aim at providing a comprehensive analysis on the cell cycle consequences associated with MLL-fusions using genetically modified cells derived from mice carrying various MLL-fusion knockin alleles, including MLL-AF4, MLL-AF9, and MLL-CBP.

Proteomics-Based Strategy to Identify Biomarkers and Pharmacological Targets in Leukemias with t(4;11) Translocations

Translocations and other aberrations involving the MLL (mixed lineage leukemia) gene result in aggressive forms of leukemias. Heterogeneity in partner genes, in chromosomal breakpoints, in MLL itself, and in the different partner genes results in heterogeneous fusion transcripts that can be alternatively spliced, which complicates deciphering a unifying mechanism of leukemogenesis. However, recent microarray studies completed with clinical leukemia specimens have uncovered several distinct mRNA signatures within MLL leukemia that differ from other types of leukemia. A global proteomics strategy using MV4-11 and RS4:11 cells in culture was employed to investigate possible protein signatures common to different MLL leukemias and to identify disease biomarkers and protein targets for pharmacological intervention. Initial proteomics screening experiments with two-dimensional differential in-gel electrophoresis revealed heat shock protein 90 alpha (HSP90alpha) as a potential target for pharmacological inhibition and nucleoside diphosphate kinase (nm23) as a biomarker for measuring treatment efficacy. Using a modified stable isotope labeling of amino acids in cell culture (SILAC) approach, coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS), changes in abundance for over 500 proteins were measured. In addition, decreased expression of the novel biomarker nm23 was observed during HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) in the MV4-11 cell line. The present study validates the use of a global proteomics strategy to uncover novel biomarkers and pharmacological targets for leukemias with MLL translocations. Additionally, several proteins were found to be expressed in concordance with microarray studies of mRNA expression in specimens from patients showing the value in comparing mRNA transcript and proteomic profiles. This work represents one of the most comprehensive proteomics screens of MLL leukemias that have been conducted to date.

MLL Translocation Partners AF4, AF5q31, and ENL Associate in a Higher Order Protein Complex with CDK9 and Cyclin T1.

The Mixed Lineage Leukemia (MLL) gene is frequently involved in chromosomal translocations that cause acute leukemia. More than 40 different genes have been identified as MLL translocation partners, with the expression of corresponding MLL fusion proteins. The MLL protein has histone methyltransferase activity and is required for embryonic development and hematopoiesis. Several proteins have been demonstrated to associate with MLL in a macromolecular complex, which is believed to have chromatin remodeling function. However, the C-terminal SET domain of MLL, which carries the histone methyltransferase activity, is lost in all MLL fusion proteins, thus making the biochemical functions of the fusion proteins unclear. Moreover, the promiscuity of MLL translocation partners, most of them with no known functions, further complicates an understanding of MLL leukemogenic mechanisms. In this study, we purified a protein complex containing AF4, the most common MLL translocation partner, using a combination of conventional column chromatography and immunoaffinity techniques. The AF4 protein complex contains AF5q31 and ENL, two other MLL translocation partners, as well as CDK9 and Cyclin T1, a heterodimer that regulates transcriptional elongation. Gel filtration confirmed that these five proteins co-fractionate with an estimated overall size of 0.8 MDa. All protein-protein interactions were further confirmed by immunoprecipitation-western blotting from K562 cell nuclear extract. To investigate whether these protein-protein interactions are retained in corresponding MLL fusion proteins, immunoprecipitation-western blotting assays were carried out in human leukemia cell lines harboring MLL chromosomal translocations. We found that MLL-AF4, MLL-AF5q31, MLL-ENL and MLL-AF9 each associate with wild type AF4 complex components, including CDK9 and Cyclin T1. In contrast, MLL-AF6 does not associate with any of the AF4 complex components. We propose that the four nuclear MLL translocation partner proteins (AF4, AF5q31, ENL/AF9), whose translocations are found in over 75% of MLL leukemias, associate in a higher order protein complex with CDK9 and Cyclin T1 and thus function in part to regulate transcriptional elongation. The association of CDK9 and Cyclin T1 with the four MLL fusion proteins suggests a common leukemogenic mechanism that may involve transcriptional elongation, which we are currently investigating. Conversely, MLL-cytosolic fusions, e.g. MLL-AF6, appear to function independently of association with the AF4 protein complex, possibly through a homo-dimerization pathway.

FLT3 Signaling Is Not Necessary to the Leukemogenic Properties of MLL Fusion Genes in a Murine Model.

The tyrosine kinase FLT3 has received much attention as a potential drug target in acute myeloid leukemias. FLT3 is expressed on hematopoietic precursors and its activation plays a role in cell survival and proliferation as well as prevention of differentiation. Activating mutations of FLT3 are found in 30% of acute myeloid leukemias (AMLs) and can be associated with an unfavorable prognosis. The majority of leukemic cells also express both FLT3 and FLT3-ligand enabling a self-activation loop.Several micro array analyses have shown that MLL leukemias, harboring a rearrangement of the Mixed Lineage Leukemia (MLL) gene on chromosome 11q23, are characterized by an exceptionally high level of FLT3 mRNA expression. Other studies have shown that FLT3 activating mutations are present in 10 to 20% of MLL leukemias. These findings suggest that the FLT3 receptor could be a valid drug target to fight these MLL leukemias against which current treatments are by enlarge inefficient.We have developed a bone marrow retroviral transduction/transplantation model to study the mechanisms of transformation by MLL fusion genes. We measured the transcript level of FLT3 using real time PCR and found that cells transformed by MLL fusion genes expressed considerably higher levels of FLT3 in vitro and in vivo compared to controls. We then investigated whether FLT3 signaling contributed to the pathogenesis of MLL leukemias in our model. We co-transduced the constitutively active FLT3ITD mutant with MLL-ENL in C57Bl6 bone marrow and compared the properties of the resulting cells in vitro and in vivo to cells solely infected with MLL-ENL. We found that FLT3ITD did not confer any advantage in terms of growth factor dependence or rate of proliferation and that the degree of myeloid maturation of the cells in culture was unaffected. Co-transduced cells inoculated to irradiated recipient mice induced acute myeloid leukemias with similar characteristics and latency as well. We next investigated if MLL-ENL could transform cells in vitro or in vivo in the absence of FLT3 signaling. Bone marrow from flt3 knock out or flt3 +/+ mice were transduced in parallel with MLL-ENL and studied in vitro and in vivo. We found that flt3−/− bone marrow could be immortalized and displayed the same properties as MLL-ENL flt3+/+ cell lines. flt3+/+ irradiated mice inoculated with flt3−/− or flt3+/+ MLL-ENL-transduced bone marrow progenitors also developed indistinguishable acute myeloid leukemias. Finally we studied the effect of the FLT3 inhibitors PKC412 (100 nM) and AG1296 (1 mM) on cells immortalized by MLL-ENL in culture. We found that both these drugs inhibited the proliferation and induced the myeloid maturation of MLL-ENL cells in culture.Surprisingly, we observed identical activities of these FLT3 inhibitors on flt3−/− cells immortalized by MLL-ENL. This can likely be accounted for by the fact that PKC412 and AG1296 are known to be inhibitors of multiple kinases, including some thought to be important for carcinogenesis. Altogether, our findings indicate that FLT3 may not be a relevant drug target for all MLL leukemias and that FLT3 inhibitors' activity in this model of MLL leukemia is mediated by interference on other signaling pathways via alternative kinase inhibition, yet to be identified.

Disease Models and Transformation Mechanisms Mediated by MLL-AF4 Family Oncoproteins in Human Leukemia.

The Mixed Lineage Leukemia (MLL) gene codes for a histone methyltransferase that is required for hematopoietic development. As a consequence of chromosomal translocations, MLL is fused with over 40 different genes to yield in-frame fusion proteins in acute leukemias. AF4, the most common fusion partner, accounts for 40% of MLL leukemias. The AF4-related proteins, LAF4 and AF5q31, are also fusion partners for MLL in rare cases of leukemia, whereas a fourth family member, FMR2, is a candidate protein for fragile X mental retardation syndrome. MLL fusions with AF4 family proteins manifest as acute biphenotypic or lymphoblastic leukemias that are associated with a poor prognosis, but the lack of appropriate disease models has hampered progress in understanding their underlying molecular mechanisms. Here we report that each of the MLL-AF4 family fusion proteins (the leukemia associated MLL-AF4, MLL-LAF4, and MLL-AF5q31 as well as an artificial MLL-FMR2) is capable of altering the growth and self-renewal properties of primary murine hematopoietic stem/progenitor cells in a serial methylcellulose culture myeloid replating assay. However, significant differences were observed in their respective oncogenic potentials. Cells transduced with MLL-LAF4, MLL-AF5q31 and MLL-FMR2 rapidly expanded during serial replatings, adapted to growth in liquid culture, and induced leukemias in syngeneic recipient mice within 6 months. Conversely, bone marrow cells transduced with MLL-AF4 yielded modest numbers of blast-like colonies in the third round of plating, only transiently expanded in vitro, and quickly underwent terminal differentiation. MLL-LAF4 and MLL-AF5q31 were also capable of enhancing the self-renewal of transduced cells with B lymphoid progenitor phenotypes. A structure/function analysis demonstrated that the previously reported transcriptional transactivation domains of AF4 family proteins were neither necessary nor sufficient for in vitro transformation. Furthermore, the recently reported AF9 interaction motif that is conserved in all AF4 family proteins was also shown to be dispensable for in vitro transformation. These data indicate that transactivation per se, and interaction with AF9 in particular, are not absolutely required for the oncogenic actions of MLL-AF4 family proteins. Conversely, a minimal transformation domain was mapped to the highly conserved carboxy-terminal homology domain shared among all AF4 family proteins, including the Drosophila homolog Lilliputian, and shown to be necessary and sufficient for transformation. Taken together, our studies establish transformation models for MLL-AF4 family fusion proteins and provide critical mechanistic insights into their underlying molecular mechanisms.

Leukemic transformation of hematopoietic progenitors by MLL-GAS7 in the absence ofHoxa7orHoxa9

AbstractDifferential expression ofHoxgenes is associated with normal hematopoiesis, whereas inappropriate maintenance ofHoxgene expression, particularlyHoxa7andHoxa9, is a feature of leukemias harboring mixed-lineage leukemia(MLL)mutations. To understand the pathogenic roles ofHoxgenes in MLL leukemias, we assessed the impact ofHoxa7orHoxa9nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguishedHoxa7-andHoxa9-deficient mice. Megakaryocytic/erythroid progenitor (MEP) reductions inHoxa7-/-mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely,Hoxa9-/-mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays,Hoxa7-andHoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However,Hoxa7-/-orHoxa9-/-progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus,Hoxa7andHoxa9contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7–mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins. (Blood. 2004;103:3192-3199)

Missense mutation and defective function of ATM in a childhood acute leukemia patient with MLL gene rearrangement

The possible involvement of germline mutation of the ataxia telangiectasia mutated (ATM) gene in childhood acute leukemia with mixed lineage leukemia (MLL) gene rearrangement (MLL(+)) was investigated. Of the 7 patients studied, 1 showed a germline missense ATM mutation (8921C>T; Pro2974Leu), located in the phosphatidylinositol-3 (PI-3) kinase domain. In reconstitution assays, the ATM mutant 8921T could only partially rescue the radiosensitive phenotype of AT fibroblasts, and in an in vitro kinase assay, it showed a defective phosphorylation of p53-Ser15. Furthermore, the introduction of 8921T in U2OS cells, characterized by a normal ATM/p53 signal transduction, caused a significant reduction of in vivo p53-Ser15 phosphorylation, suggesting a dominant-negative effect of the mutant ATM over the wild-type protein. Our finding in this patient suggests that altered function of ATM plays some pathogenic roles in the development of MLL(+) leukemia.