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

Abstract

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.

Fig 1. NUP98-HOXA9 expression disrupts Drosophila eye development.

(A) Schematic representation of the NUP98-HOXA9 (NA9) variants used in this study. Full-length NUP98 and HOXA9 proteins are shown as reference. The W506A mutation in the PBX-Interaction Motif (PIM) abrogates PBX binding. The N562A mutation in the Homeodomain (HD) prevents DNA-binding. (B-G and I-K) Micrographs of representative adult Drosophila eyes expressing one copy of the indicated transgenes under the UAS promoter driven by ey-Gal4. The LacZ transgene is used as control. (H and L) Eye size quantification of the indicated genotypes. Statistical significance was assessed using a Student’s t test. Posterior is to the left and dorsal is up. The same orientation is used for all adult eyes and eye imaginal discs shown throughout this study.

Fig 2. NUP98-HOXA9 collaborates with HTH during Drosophila eye development.

FLP-out clones produced in the eye using ey-FLP; Act5C > CD8 > Gal4, UAS-GFP were analyzed at the adult and larval stages. (A, C, E, G) Micrographs of representative adult Drosophila eyes clonally expressing (A) UAS-LacZ/+, (B) UAS-NA9/+, (C) UAS-GFP-hth, and (D) UAS-NA9/UAS-GFP-hth as indicated to the left of each panel. (B, D, F, H) Third instar eye discs labeled with BrdU revealing cells in S phase. GFP staining marks the positions of clones expressing the transgenes indicated to the left of the panels. Yellow arrowhead indicates the position of the second mitotic wave. (I) Quantification of eye size and tissue overgrowth. Statistical significance was assessed using a Student’s t test.

Fig 3. Examples of modifiers recovered in the NA9 screen.

(A-E) Micrographs of representative adult Drosophila eyes of the indicated genotypes. Shown to the right are the corresponding relative distribution of eye modifications compared to the baseline phenotype induced by NA9 expression. Note that the overall phenotype of the control CNA9/+ genotype shows some variations mostly in the direction of mild suppression. Modifiers were scored as weak (W), medium (M) or strong (S) Enhancers (E) or Suppressors (S). At least 30 flies were scored for each cross presented.

Fig 4. Molecular lesions associated with eight modifiers of NA9.

(A) Schematic representations of proteins encoded by loci identified in the screen. Domain compositions were defined according to the Conserved Domains tool at the National Center for Biotechnology Information (NCBI)). The position of amino acid changes for the indicated sequenced alleles is shown on top of each protein schematics. Asterisks denote STOP codons. (B-E) Micrographs of adult eyes of the following genotypes: (B, C) Oregon R flies denoted as WT and (D, E) CNA9/+ denoted as CNA9. Developing flies were treated with DMSO (B and D) or selinexor (C and E). Mean eye size (expressed as percent compared to WT), is indicated at the bottom right of CNA9 panels. The experiment was conducted in triplicate and at least five eyes have been analyzed per condition. Statistical significance (*; p≤4.2E-07) of eye size difference between DMSO versus selinexor-treated CNA9 flies (D, E) was assessed using a Student’s t test.

Fig 5. Knockdown of candidate genes by RNAi modulates the NA9 phenotype in a manner similar to mutant alleles.

Micrographs of adult Drosophila eyes of the following genotypes: (A) ey-Gal4, UAS-NA9/+ (NA9). (B-L) ey-Gal4, UAS-NA9/UAS-dsRNA lines as indicated. Yellow arrows point to wing-like structures emerging mostly from the anterior portion of adult eyes.

Fig 6. E(Pc) inactivation enhances NA9-induced eye-to-wing transdetermination.

(A, C, E, G, I, K) Micrographs of adult Drosophila eyes. (B, D, F, H, J, L) Third instar larval eye discs immunostained with anti-Vestigial (Vg) and anti-Eyes Absent (Eya) antibodies. DAPI staining marks the nuclei. The following genotypes were analyzed: (A, B) ey-Gal4/+ referred to as WT. (C, D) ey-Gal4/E(Pc)^E-4112. (E, F) ey-Gal4/UAS-E(Pc)^dsRNA. (G, H) ey-Gal4, UAS-NA9/+. (I, J) ey-Gal4, UAS-NA9/E(Pc)^E-4112. (K, L) ey-Gal4, UAS-NA9/UAS-E(Pc)^dsRNA. Proportion (%) of eyes exhibiting ectopic wing material is indicated at the bottom right of each eye micrograph. Quantifications are shown in Table 2. Yellow arrow points to the anterior dorsal margin exhibiting Vg expression.

Fig 7. Drosophila CBP/p300 and HDAC1 activity influence NA9-mediated transdetermination.

(A, C, E, G, I) Micrographs of adult Drosophila eyes. (B, D, F, H, J) Third instar larval eye discs immunostained with anti-Vestigial (Vg) and anti-Eyes Absent (Eya) antibodies. DAPI staining marks the nuclei. The following genotypes were analyzed: (A, B) ey-Gal4, UAS-NA9/+. (C, D) nej³/+; ey-Gal4, UAS-NA9/+. (E, F) ey-Gal4, UAS-NA9/+; UAS-Nej-V5/+. (G, H) ey-Gal4, UAS-NA9/UAS-HDAC1^dsRNA. (I, J) ey-Gal4, UAS-NA9/+; UAS-V5-HDAC1/+. Proportion (%) of eyes exhibiting ectopic wing material is indicated at the bottom right of each eye micrograph. Quantifications are shown in Table 2. Yellow arrow points to the anterior dorsal margin exhibiting Vg expression.