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By Harish G. Ahuja, MD
Non-random chromosomal translocations such as translocations, deletions, and inversions frequently are associated with a wide variety of hematological malignancies and generally are thought to be causal events in the process of leukemic transformation.1 In most instances, chromosomal translocations fuse sequences of a transcription factor, transcription modulator, or receptor tyrosine kinase to those of a normally unrelated gene, resulting in a chimeric protein with oncogenic properties.1
Repositioning of transcriptional control genes in the vicinity of highly active promoter/enhancer elements, such as those associated with the immunoglobulin or T-cell receptor genes, is a second mechanism by which translocations induce malignancy.1 Similar chromosomal translocations also are associated with therapy-related myelodysplastic syndromes and/or acute myelogenous leukemia (t-MDS/AML) and are thought to arise as a result of exposure to various forms of chemotherapy.2 Most cases of t-MDS/AML arising after exposure to alkylating agents are associated with loss of chromosomes 5 and/or 7 or various parts of the long arm of these chromosomes.2 On the other hand, agents that target the enzyme topoisomerase II (topII), such as epipodophyllotoxins or anthracyclines, cause leukemia characterized by a short latency period, monocytoid features, and usually balanced translocations involving the MLL gene on chromosome 11q23.3,4 Other balanced translocations, e.g., t(8;21), t(15;17), and inv(16), which characteristically occur in de novo AML have been reported, albeit infrequently, in t-MDS/AML as well.4-6
The NUP98 Gene
The NUP98 gene on chromosome 11p15 is a 98 KD component of the nuclear pore complex that is presumed to function as a docking protein controlling nucleocytoplasmic transport.7 This docking function is mediated by multiple FXFG repeats located in the N-terminal portion of the gene.7 Several recent reports have indicated that the NUP98 gene is found at the breakpoints of several distinct chromosomal breakpoints in human hematological malignancies. These include: t(7;11)(p15;p15), t(2;11)(q31;p15), t(1;11)(q23;p15), inv(16)(p15q22), and t(4;11)(q21;p15). Chimeric mRNAs spliced across the breakpoints fuse the FXFG repeats of NUP98 with HOX A9, HOX D13, PMX-1 (all homeodomain-containing proteins), DDX 10 (a putative RNA helicase), and RAP1GDS1 (a guanine nucleotide exchange factor).8-13 Significantly, with the exception of the t(4;11) which has been reported in a patient with T cell acute lymphoblastic leukemia, all of the aforementioned translocations have been reported in patients with t-MDS/AML, arising in the context of exposure to multi-agent chemotherapy that has included a topII inhibitor.
The t(11;20)(p15;q11.2) is a rare but recurrent chromosomal translocation that has been reported in patients with MDS, AML, and polycythemia rubra vera.14-16 We identified two children who developed t-MDS/AML associated with the t(11;20) following exposure to multi-agent chemotherapy that included a topII poison.14 Using 3’ RACE we have cloned the fusion transcript arising as a result of this translocation and have demonstrated an in-frame fusion between 5’ NUP98 FXFG repeats and the main body of DNA topoisomerase 1 (topI).17 TopI normally catalyses a series of transesterification reactions during which it generates transient single-stranded DNA breaks that result in topological transformations in DNA.18 TopI also has been shown to be involved in DNA replication, transcription, and recombination, as well as chromosome condensation.18 The topI protein can be organized into four distinct domains: a C-terminal catalytic region that contains the active tyrosine; a non-conserved linker domain; a conserved "core" domain; and a N-terminal region that contains a nuclear localization signal.18 Although it has been shown that this N-terminal region is dispensable for catalytic activity, several potentially significant interactions involving this region of topI have been reported. These include interactions with nucleolin, SV40 large T antigen, and the SF2/ASF splicing factor.19-21 The NUP98-topI fusion protein lacks this N-terminal region and it is possible that loss of regulatory functions dependent on this N-terminal region may contribute to leukemogenesis. It also has been recently been shown that topI interacts with p53.22 It is conceivable that loss of this interaction as a result of the translocation also could contribute to leukemogenesis. Finally, it has been demonstrated that the NUP98-HOX A9 fusion protein can transform NIH 3T3 fibroblasts in vitro.23 The mechanism suggested is one in which HOX A9-responsive genes are activated by the transactivating properties of the NUP98 FXFG repeats. A similar mechanism could explain the potential leukemogenicity of the NUP98-topI fusion.
To investigate potential mechanisms responsible for the generation of the t(11;20) associated with t-MDS/AML, we cloned and sequenced the genomic breakpoints from the two patient samples.24 Nucleotide sequence analysis of the germline as well as rearranged alleles revealed almost perfectly balanced translocations with no net gain or loss of DNA. We did not detect any known recombinogenic sequences such as Alu repeats, purine/pyramidine repeat regions, palindromi sequences, consensus x-like sequences, heptamer-nonamer se-quences, or putative topII consensus recognition sites at or near the breakpoints. However, close analysis of the breakpoint junction sequences revealed four nucleotide microduplications at the breakpoint junctions. The simplest hypothesis to explain the four nucleotide microduplications at the breakpoint junctions proposes that the translocations were generated by four nucleotide staggered double-stranded DNA breaks, allowing the four nucleotide overhang to serve as a template on each derivative chromosome. Given the history of exposure to topII poisons and the fact that such drugs stabilize the staggered breaks when topII binds covalently to DNA, it seems plausible that topII acted as a catalyst in inducing these rearrangements perhaps through an exchange of topII subunits. Taken together with previous reports of NUP98 rearrangements in patients with t-MDS/AML, our findings suggest that NUP98, like MLL, is a frequent target for chromosomal translocations in patients with t-MDS/AML that develops after exposure to multi-agent chemotherapy containing a topII poison.
NUP98 rearrangements are not restricted to patients with t-MDS/AML and occur in patients with de novo AML as well. We recently have reported the cloning of a t(9;11)(p22;p15) from a patient with de novo AML and have demonstrated that the translocation results in a fusion between the NUP98 gene and the gene encoding the transcriptional co-activators p52 and p75, both of which are derived through alternative splicing from a single gene known as lens epithelium-derived growth factor (LEDGF).25 Both NUP98-p52 and NUP98-p75 chimeric mRNAs were detectable in the patient sample. The p52 and p75 proteins are homologous to the hepatoma-derived growth factor (HDGF) and the HDGF-related proteins 1 and 2 (HRP-1 and HRP-2).26 The highest degree of homology (80%) is found in the N-terminal amino acid residues also known as the HATH region (homologous to the N-terminus of HDGF) and it is this region that is lost from the fusion protein. The C-terminal region shows similarity to HMG-1, a multifunctional non-histone protein involved in many steps of gene regulation. p52 is a potent transcriptional co-activator and is thought to mediate functional interactions between upstream sequence-specific activators and the general transcriptional apparatus. It also has been shown to interact with the essential splicing factor ASF/SF2 to modulate mRNA splicing.26 p75 is a less potent co-activator than p52 and does not interact functionally with ASF/SF2. However, it has been shown to function as a growth and survival factor for lens epithelial cells, keratinocytes, and skin fibroblasts.27
It is intriguing that two fusion proteins with potentially differing functions were detectable. The relative contribution of each of these fusion proteins to leukemogenesis is under study.
The exact mechanism whereby these NUP98 fusions cause leukemia remains speculative. One possible scenario is the disruption of functions normally attributable to NUP98 or the various partner genes. Another scenario is through a "gain of function" acquired as a result of fusion to the NUP98 FXFG repeats. Future experiments in which NUP98 or the various partner genes are mutated and homozygously inactivated in "knock-out" mice or transgenic mouse models in which the various fusion genes are overexpressed will undoubtedly shed light into some of these mechanisms. (Dr. Ahuja is a Medical Oncologist at the University of Wisconsin Comprehensive Cancer Center in Wausau.)
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