(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene

['Gwenaëlle Gavory', 'Institute For Research In Immunology', 'Cancer', 'Université De Montréal', 'Montréal', 'Caroline Baril', 'Gino Laberge', 'Gawa Bidla', 'Surapong Koonpaew', 'Thomas Sonea']

Date: 2021-10

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

Acute myeloid leukemia or AML is a cancer of blood cells. Despite significant progress in recent years, a majority of afflicted individuals still succumbs to the disease. A variety of genetic defects have been associated to AML. Among these are chromosomal translocations, which entail the fusion of two genes, leading to the production of cancer-inducing chimeric proteins. A representative example is the NUP98-HOXA9 oncoprotein, which results from the fusion of the NUP98 and HOXA9 genes. The mechanism of action of NUP98-HOXA9 remains poorly understood. Given the evolutionarily conservation of NUP98 and HOXA9 as well as basic cellular processes across multicellular organisms, we took advantage of Drosophila fruit flies as a genetic tool to identify genes that impinge on the activity of human NUP98-HOXA9. Surprisingly, this approach identified a relatively large spectrum of conserved genes that engaged in functional interplay with NUP98-HOXA9, which indicated the pervasive effects that this oncogene has on basic cellular events. While some genes have been previously linked to NUP98-HOXA9, thus validating our experimental approach, several others are novel and as such represent potentially new avenues for therapeutic intervention.

Funding: G.G. was recipient of a doctoral studentship from Fonds de recherche du Québec - Santé ( http://www.frqs.gouv.qc.ca/ ). M.T. holds a Canada Research Chair in Intracellular Signalling ( https://www.chairs-chaires.gc.ca/ ). This work was supported by operating grants from the Canadian Institutes of Health Research ( https://cihr-irsc.gc.ca/ ) (MOP93654) and the Leukemia & Lymphoma Society of Canada ( https://www.llscanada.org/ ) to M.T and G.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Gavory et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we show that expression of human NA9 during Drosophila eye development induces a phenotype that relies on the same functional elements as those originally defined in mammals. We exploited this system in a modifier screen to isolate genetic modulators of NA9 activity. This approach uncovered 29 complementation groups of mutations that dominantly alter the NA9 eye phenotype. Of these groups, three correspond to genes (Rae1, emb and hth) that have homologs in mammals (RAE1, XPO1 and MEIS1/2) that have previously been reported to influence the leukemogenic activity of NA9 [ 26 , 29 , 31 , 41 , 42 ]. Interestingly, the screen uncovered evolutionarily-conserved genes encoding a variety of functions, such as chromatin remodeling, nuclear export, cell polarity, cytoskeletal organization and translation, suggesting that NA9 impinges on a multiplicity of cellular processes. Unexpectedly, a characterization of the NA9-induced eye phenotype revealed that it is largely based on the transdetermination of eye cells into wing cells and that several genetic modifiers of NA9 influence this activity. Together, this study identifies NA9 as a disruptor of epigenetic regulation and unveils a large cohort of modifiers that might prove critical for its leukemia-inducing property.

Since not required for viability or fertility, Drosophila eyes are particularly well suited for interrogating complex biological events by genetic means. Their use have indeed led to the discovery of numerous signaling mechanisms and developmental processes conserved across metazoans [ 37 – 39 ]. Emerging from an epithelium known as the eye-antennal imaginal disc, the Drosophila eye is a highly organized structure composed of about 800 photosensitive units called ommatidia [ 40 ]. Since closely related NUP98 and HOXA9 homologs are present in Drosophila (known as NUP98 and Abd-A/B, respectively), we reasoned that expression of human NA9 in fly eyes could disrupt protein networks related to those perturbed in mammalian cells and thus produce phenotypes representative of NA9 function amenable to genetic screening.

A variety of models have been used to characterize the molecular and cellular events underlying the leukemogenic activity of NA9 [ 26 , 28 , 35 , 36 ]. For example, we have recently shown that expression of human NA9 in the hematopoietic organ of Drosophila larvae, called the lymph gland, triggers the premature differentiation of hemocyte progenitors followed by their proliferation [ 35 ]. This work revealed a need for the same functional elements as those originally delineated using mammalian models. We concluded that Drosophila could represent a relevant genetic system to identify molecular events impaired by NA9.

Insights into NA9 activity was originally acquired by conducting structure-function analysis experiments in mammalian cells and in vivo mouse models of AML [ 27 , 28 ]. These studies suggested that NA9 acts as an aberrant transcription factor whereby the homeodomain binds to DNA and the NUP98 moiety serves as a transcriptional activation domain. NUP98 FG repeats appear to influence transcription in part through physical interactions with the transcriptional co-activators CREB-binding protein (CBP) and p300, which are histone acetyltransferases (HAT), and with the transcriptional co-repressor HDAC1, a histone deacetylase [ 27 , 28 ]. NA9 also likely perturbs nucleocytoplasmic trafficking by sequestering the nuclear export factors RAE1 and Exportin1 (XPO1)/CRM1 by association with the GLEBS domain and FG repeats of the NUP98 moiety [ 29 , 30 ]. Conversely, chromatin-bound XPO1 was recently found to recruit NA9 to HOX genes and induce their expression [ 31 ]. MLL1 was shown to contribute to the oncogenic properties NA9 by recruiting NA9 to the HOXA/B locus via an interaction with the FG repeats of the NUP98 portion, thereby inducing HOXA/B gene expression [ 32 , 33 ]. Interestingly, a recent BioID screen conducted in the colon cancer cell line HCT-116, identified XPO1, RAE1, HDAC1 and MLL1 as proximal interactors of a NA9-BirA bait [ 34 ].

Several recurrent chromosomal translocations also promote AML [ 23 ]. Among these, a diverse set of fusions involving nucleoporin genes have been detected, where NUP98 is the most frequently affected gene [ 8 ]. The prototype chimera is NUP98-HOXA9 (referred hereafter to NA9), which encodes the N-terminal Phe-Gly (FG)-rich repeat portion of NUP98 fused to the C-terminal portion of HOXA9 that comprises a PBX-Interacting Motif (PIM) and a DNA-binding homeodomain [ 24 , 25 ]. Mice transplanted with NA9-expressing bone marrow cells develop a myeloproliferative disease that ultimately progresses to AML after a long latency and is accompanied by an upregulation of the HOX loci [ 26 ]. As with HOXA9, NA9-induced leukemia is accelerated by co-expression of MEIS1 [ 26 ].

Homeobox (HOX) genes encode homeodomain-containing transcription factors that are the main regulators of mammalian development [ 7 ]. They also play essential roles in hematopoiesis throughout development and adult life [ 8 ]. Dysregulation of HOX gene expression in hematopoietic stem cells is closely associated to AML, which appears to be a common and cooperative event with driver mutations in genes such as NPM1, FLT3 and IDH1/2 [ 9 – 11 ]. A typical example is HOXA9, which is overexpressed in more than 50% of AML cases and has been defined as the most predictive marker of poor prognosis of AML [ 12 , 13 ]. Multiple mechanisms control the expression of HOXA9 and their perturbations also lead to the development of AML. Among the clearest examples are the epigenetic regulators Mixed Lineage Leukemia (MLL; a histone H3K4 methyltransferase) and Polycomb Repressive Complex 2 (PRC2; harbors H3K27 methyltransferase activity) that positively and negatively regulate, respectively, the transcriptional activity HOXA9 [ 14 – 16 ]. Aberrant activation of MLL by chromosomal translocations or inactivation of PRC2 subunits by loss-of-function mutations or silencing are conducive to AML onset and these genetic lesions are frequently accompanied by the upregulation of HOXA9 expression [ 17 ]. Consistent with the relevance of HOXA9 in AML, its forced expression in murine bone marrow cells produces a preleukemic phase which, after a long latency, develops into full-fledged AML [ 18 ]. This last observation suggested early on the involvement of secondary collaborative events. These include the co-expression of the TALE (three amino-acid loop extension) family of co-factors, MEIS and PBX, which increase the DNA-binding affinity and specificity of HOX proteins [ 19 – 21 ] and significantly accelerate the onset of HOX-mediated AML [ 18 , 22 ].

Acute myeloid leukemia (AML) is among the most common and deadliest forms of leukemia affecting the ageing human population [ 1 ]. It is a clonal disease of hematopoietic stem cells characterized by the interruption of myeloid differentiation and the relentless proliferation of abnormal progenitors accumulating in bone marrow, blood and other tissues. Molecular lesions impinging on relatively few genes have been linked to the pathogenesis of AML [ 2 ]. DNMT3A, FLT3, IDH1, IDH2, and NPM1 are among the most commonly mutated loci [ 3 , 4 ]. Recent advances in genomics is improving patient stratification, allowing for better therapeutic regimens [ 5 , 6 ]. However, the prognosis remains bleak and a deeper understanding of the underlying mechanistic causes of AML remains absolutely necessary to accelerate the development of effective therapies.

Results

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009730

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.