Search

Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extra-terminal motif (BET) protein family (BRD2, BRD3 and BRD4). Introduction of the W390A mutation in MLV IN abolishes BET interaction. Here we compared the replication of W390A MLV and WT MLV in adult BALB/c mice to study the role of BET proteins in replication, integration and tumorigenesis in vivo. Comparing WT and W390A MLV infection revealed similar viral loads in blood, thymus and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV and W390A MLV developed T cell lymphoma after a similar latency represented by an enlarged thymus and spleen and multi-organ tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profile of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters but compared to WT, W390A MLV integrated less frequently into enhancers and more into oncogene bodies, such as Notch1 and Ppp1r16b. We conclude that host factors direct MLV in vivo integration site selection. Although, BET proteins target WT MLV integration preferentially towards enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV LTR.

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival. ispartof: CELLS vol:10 issue:1 ispartof: location:Switzerland status: published

Mixed lineage leukemia (MLL) fusion–driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium–derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75–MLL–MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75–MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors—HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL–AF9+ leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9–expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75–HIV-1 integrase interaction. The newly defined protein–protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75–dependent MLL fusion–driven leukemic disorders. Cancer Res; 74(18); 5139–51. ©2014 AACR.

Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV INW390A efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34+ hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy. publisher: Elsevier articletitle: Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy journaltitle: Molecular Therapy - Nucleic Acids articlelink: http://dx.doi.org/10.1016/j.omtn.2017.04.002 content_type: article copyright: © 2017 The Authors. ispartof: Molecular Therapy-Nucleic Acids vol:7 pages:231-245 ispartof: location:United States status: published

Mixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of human acute leukemia carrying chromosomal translocations of the MLL gene. These translocations result in oncogenic fusions that mediate aberrant recruitment of the transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form a complex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both MENIN and LEDGF/p75 are required for efficient MLL-fusion-mediated transformation and for the expression of downstream MLL-regulated genes such as HOXA9 and MEIS1 In light of developing a therapeutic strategy targeting this complex, understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockout mouse model in the hematopoietic compartment and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem cells, and decreased MLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation of MLL-mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia.

Stable integration in the host genome renders murine leukemia virus (MLV)-derived vectors attractive tools for gene therapy. Adverse events in otherwise successful clinical trials caused by proto-oncogene activation due to vector integration hamper their application. MLV and MLV-based vectors integrate near strong enhancers, active promoters, and transcription start sites (TSS) through specific interaction of MLV integrase (IN) with the bromodomain and extra-terminal (BET) family of proteins, accounting for insertional mutagenesis. We identified a BET-interaction motif in the C-terminal tail of MLV IN conserved among gammaretroviruses. By deletion of this motif or a single point mutation (INW390A), BET-independent MLV (BinMLV) were engineered. BinMLV vectors carrying INW390A integrate at wild-type efficiency, with an integration profile that no longer correlates with BET chromatin distribution nor with the traditional markers of MLV integration. In particular, BinMLV vector integration associated less with oncogene TSS compared to the MLV vectors currently used in clinical trials. Together, these findings open perspectives to increase the biosafety of gammaretroviral vectors for gene therapy. ispartof: Molecular therapy. Nucleic acids vol:3 issue:7 ispartof: location:United States status: published

A hallmark of retroviral replication is integration of the viral genome into host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to murine leukemia virus (MLV)-derived vector integration, hamper their application. Here, we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3, and BRD4) and MLV integrase specifically interact and colocalize within the nucleus of the cell. Inhibition of the BET proteins' chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75 retargets MLV integration away from transcription start sites and into the body of actively transcribed genes, conforming to the HIV integration pattern. Together, these data validate BET proteins as MLV integration targeting factors. publisher: Elsevier articletitle: The BET Family of Proteins Targets Moloney Murine Leukemia Virus Integration near Transcription Start Sites journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2013.09.040 content_type: article copyright: Copyright © 2013 The Authors. Published by Elsevier Inc. ispartof: CELL REPORTS vol:5 issue:4 pages:886-894 ispartof: location:United States status: published

Rearrangements of the MLL (mixed lineage leukemia) gene are associated with pediatric, adult and therapy-related acute leukemias of both the myeloid and lymphoid lineage. Today, about 60 MLL fusion partner genes have been characterized at the molecular level involving almost all chromosomes. Nearly one-third of these partner genes have been identified at the DCAL (Diagnostic Center of Acute Leukemia) where more than 700 MLL rearrangements, including 36 different partner genes, have been analyzed so far. 85% of the analyzed MLL rearrangements account for the 6 most frequent partner genes, and 15% for less frequent partner genes including the 8 novel partner genes ACTN4, C2CD3, DCP1A, FLNA, LAMC3, LOC100128568, NRIP3, and TNRC18. Here, we present an overview of all current known translocation partner genes. This overview indicates all the specific introns of the TPGs found to be involved in MLL translocations, their chromosomal locations and the type of genetic abnormality that is leading to the MLL fusion. Additionally, the current breakpoint cluster region (bcr) is also presented for the 10 most frequent partner genes, namely AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10, MLLT4 (AF6), ELL, EPS15, MLLT6 (AF17), MLLT11 (AF1Q) and partial tandem duplications (PTDs). These bcrs have been used as basis for the development of an long range multiplex PCR. This is an additional tool for the identification of genomic MLL breakpoints to enhance the diagnostic efficiency. Approximately 20% of all MLL rearrangements are complex ones. In this study, we have identified 109 (15%) complex rearrangements. Within these complex rearrangements, the 5’ part of the MLL gene is generally fused in frame to one of the most frequent partner genes. But the 3’ part of the MLL gene is fused in 45 cases (41%) to a novel so-called “reciprocal MLL partner gene” like ADSS or ATG16L2. This heterogeneous group of novel translocation partner genes had not been identified as fusion partners to the 5’ part of the MLL gene before. Whether the pathobiology of complex rearrangements is different in comparison to their not complex counterparts has still to be proven. Also a novel “spliced MLL fusion” involving the AFF1 gene has been identified at the DCAL. In this case, the breakpoint is 90 kb up-stream of the AFF1 gene. Unfortunately, no cDNA/RNA was left to identify the chimeric fusion transcript. Nevertheless, the disease phenotype of a biphenotypic AL strengthens the involvement of the AFF1 gene. With these results, the number of genes involved in „ spliced MLL fusions“ has increased from seven to eight. For five patients, no partner gene could be identified at the molecular level. Also the “spliced MLL fusions” which is a mechanism to generate functional chimeric fusion transcripts with neighbouring genes described already for 8 partner genes of the MLL gene, could not be identified for these patients by RACE and RT-PCR. Furthermore, the determined patient-specific fusion sequences are succesfully used worldwide for minimal residual disease (MRD) monitoring to improve the treatment and outcome of acute leukemia patients.