Ann Lab Med.  2016 Mar;36(2):85-100. 10.3343/alm.2016.36.2.85.

Systematic Classification of Mixed-Lineage Leukemia Fusion Partners Predicts Additional Cancer Pathways

• Marschalek R

• KMID: 2373507
• DOI: http://doi.org/10.3343/alm.2016.36.2.85

Abstract

Chromosomal translocations of the human mixed-lineage leukemia (MLL) gene have been analyzed for more than 20 yr at the molecular level. So far, we have collected about 80 direct MLL fusions (MLL-X alleles) and about 120 reciprocal MLL fusions (X-MLL alleles). The reason for the higher amount of reciprocal MLL fusions is that the excess is caused by 3-way translocations with known direct fusion partners. This review is aiming to propose a solution for an obvious problem, namely why so many and completely different MLL fusion alleles are always leading to the same leukemia phenotypes (ALL, AML, or MLL). This review is aiming to explain the molecular consequences of MLL translocations, and secondly, the contribution of the different fusion partners. A new hypothesis will be posed that can be used for future research, aiming to find new avenues for the treatment of this particular leukemia entity.

Keyword

MLL-r leukemia; Translocation partner genes; Molecular mechanisms of cancer

MeSH Terms

Alleles
Chromosomes, Human, X
Epigenesis, Genetic
Humans
Leukemia/classification/*genetics/pathology
Myeloid-Lymphoid Leukemia Protein/chemistry/genetics
Protein Structure, Tertiary
Translocation, Genetic
Myeloid-Lymphoid Leukemia Protein

Figure

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  Fig. 1 Frequency of diagnostic fusion gene detection in mixed-lineage leukemia-rearranged (MLL-r) acute leukemia. Both charts summarize our knowledge about the incidence of MLL fusion partner genes that have been diagnosed at the molecular level from 1,557 acute leukemia patients. The investigated cohort was separated by disease phenotype (ALL or AML), while all others (MLL or other disease phenoytpe) were not included here. The most frequent fusion partners are depicted by black numbers for ALL (90%: AF4, ENL, and AF9) and AML patients (76%: AF9, AF10, ELL, AF6, and MLL PTDs). The remaining portions (10% for ALL, 24% for AML) represent all other yet identified MLL fusions.

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  Fig. 2 Known MLL binding proteins and functional domains. A full-length MLL protein is depicted (amino acid 1-4,005). The exon-structure (1-37) is shown above the protein structure, and the major breakpoint cluster region (BRX) comprizing introns 9-11 was depicted. All known protein binding partners (top) as well as all characterized domains with their associated functions (bottom) are indicated. FYRN and FYRC are dimerization domains that are used after Taspase1 cleavage to assemble the MLL backbone (green dotted line) for further complex formation with its binding partners. The MLL complex has epigenetic reading and writing functions and binds predominantly in the promoter regions of actively transcribing genes. A fully assembled MLL complex is shown in the bottom right.

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  Fig. 3 The PHD1-3-BD-PHD4 domain of MLL and its associated functions. (A) A portion of the MLL protein is displayed (amino acid 1,180-2,013). It starts with the MBD domain of MLL (amino acid 1,180-1,227) and ends after ePHD4 subdomain (amino acid 1,909-2,013). PHD subdomains are depicted as zinc cluster domains, and the distances between the single cysteine residues are depicted by numbers. The extended PHD subdomains (ePHD3 and 4) are composed of a normal zinc finger and a PHD subdomain. Inbetween ePHD3 and ePHD4 is a non-functional Bromo domain localized (BD: amino acid 1,669-1,803). The BD is necesary for PHD3 in order to function as H3K4me2/3 reader domain. The BD together with ePHD4 is required to bind to the ECSASB2 protein that causes the proteasomal degradation of MLL. The PHD2 subdomain is required for the dimerization of MLL protein. (B) Binding of CYP33 to ePHD3 switches this function off and enables binding of BMI1/HCP2/HDAC1/2 to the MBD domain.

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  Fig. 4 Proposed model for the oncogenic conversion of MLL fusions. (A) Physiological situation of MLL functions. Taspase1 cleaved MLL is assembled into the holo-complex and binds to target promoter regions. This occurs via the N-terminally bound MEN1/LEDGFprotein complex that allows binding to many transcription factors. The PHD domain is able to read histone core particles, while the SET domain allows writing epigenetic signatures (H3K4me2/3). Associated CREBP and MOF are able to acetylate nucleosomes. CYP33 allows switching into the "repressor mode" by enabling the docking of a Polycomb group complex composed of BMI1, HPC2, CtBP, and several HDACs. This enables to remove acetyl groups from nucleosomes or transcription factors in order to shut down gene transcription. (B) In case of a chromosomal translocation, the intrinsic regulatory mechanism of MLL becomes destroyed. The disrupted MLL portions are fused to protein sequences deriving from a large amount of different partner genes (n=82). The N-terminal portion of MLL retains the ability to bind MEN1 and LEDGF, and thus, to bind to target promoter regions. Depending on the fusion sequence (AF4, AF5, LAF4, AF9, ENL, AF10), MLL-X fusions may recruit the endogenous AF4 complex that contains P-TEFb and the histone methyltransferases DOT1L, NSD1 and CARM1, respectively. This enhances strongly transcriptional processes and results in enhanced epigenetic signatures (H3K79me2/3). However, the interactome of all other fusion sequences is not yet investigated. The C-terminal portion retains CREBBP and MOF binding capacity, as well as the SET domain. In some cases (AF4, AF5, LAF4), the N-terminal fused protein sequences allow to bind P-TEFb and directly to the largest subunit of RNA polymerase II in order to enhance the process of transcriptional elongation. In addition, the fused protein sequences still bind NSD1 and DOT1L. Therefore, the transcribed gene region aquires a highly unusual histone signature (H3K79me2/3, H3K36me2, and HeK4me2/3). This results in promoter-like signatures in the transcribed gene bodies, which in turn may help to reactivate neighboring genes over time.

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  Fig. 5 New classification of known MLL fusion genes. All known 82 fusion partner genes were classified according to their intracellular localization and function. The following pathways were assigned to the 59 cytosolic proteins: A. endocytosis and vesicle trafficking (n=9); B. FAP-mediated SRC/RAC/RHO signaling (n=18); C. ABL/other signaling pathways (n=5); D. extracellular matrix (n=1); E. non-classifiable (n=4); F. other processes (n=6); G. mitochondrial matrix protein (n=1); H. RNA decay (n=4); I. Metabolism (n=2); J. Microtubuli 6 cytokinesis associated signaling (n=9). The following pathways were assigned to the 23 nuclear proteins: K. Apoptosis (n=2); L. Centrosome & spindle apparatus (n=3); M. DNA & Chromatin (n=4); N. Signaling targets (n=6); O. Transcriptional elongation (n=8).

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  Fig. 6 Functional association of 28 MLL fusion partner proteins with 2 functional protein networks. Many MLL fusion partner proteins are ubiquitinated and subject for Ub-recognizing proteins. This allows these MLL fusions proteins to interact either with the UBC-TSG101-HGS and the UBC-HSP90AA1-PCNA-UBE2N-UBE2K-POLH-RPS3-PSMC2-PSMD4 protein network. The consequences and pathways of this protein-interaction network are depicted and are mostly occuring in the cytosol. Only a few proteins act in the nucleus (white text on blue sectors).

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  Fig. 7 TSG101 reporter cell lines to investigate protein delocalization. (A) The UBC-TSG101-HGS pathway is involved in endosomal sorting and exosome formation by linking mono-Ubc proteins to the ESCRT system. Mono-Ubc MLL fusion proteins may therefore lead to a translocation of TSG101 into the nucleus. This may cause a downregulation of p53 and a block of p21 transcription which results e.g. in enhanced growth properties. (B) First experiments with TSG101 sensor proteins (TSG101::mCh and TSG101::GFP) and co-transfected MLL fusions. Stable cell lines were selected and the expression of all four MLL fusion proteins was induced by Doxyxcycline for 48 hr. Translocations of cytosolic TSG101 into the nucleus was observed upon transfection with different MLL fusions (mCh::MLL-SMAP1, mCh::MLL-LASP1, GFP::SMAP1-MLL); solely GFP::LASP1-MLL remained in the golgi apparatus, however, was colocalizing again with the corresponding sensor protein.

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