A novel NUP98-JADE2 fusion in a patient with acute myeloid leukemia resembling acute promyelocytic leukemia

Abstract

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia (AML) characterized by block of differentiation at the promyelocytic stage and the presence of PML-RARA fusion. In rare instances, RARA is fused with other partners in variant APL. More infrequently, non-RARA genes are rearranged in AML patients resembling APL. However, the underlying disease pathogenesis in these atypical cases is largely unknown. Here, we report the identification and characterization of a NUP98- JADE2 fusion in a pediatric AML patient showing APL-like morphology and immunophenotype. Mechanistically, we showed that NUP98-JADE2 could impair all-trans retinoic acid (ATRA)-mediated transcriptional control and myeloid differentiation. Intriguingly, NUP98-JADE2 was found to alter the subcellular distribution of wild-type JADE2, whose down-regulation similarly led to attenuated ATRA-induced responses and myeloid activation, suggesting that NUP98-JADE2 may mediate JADE2 inhibition. To our knowledge, this is the first report of a NUP98-non-RAR rearrangement identified in an AML patient mimicking APL. Our findings suggest JADE2 as a novel myeloid player involved in retinoic acid-induced differentiation. Despite lacking a rearranged RARA, our findings implicate that altered retinoic acid signaling by JADE2 disruption may underlie the APL-like features in our case, corroborating the importance of this signaling in APL pathogenesis.

Figure 1.

Identification of the NUP98-JADE2 fusion. (A) Left, May-Grünwald-Giemsa staining of leukemic blasts in the diagnostic bone marrow (BM). The blasts were medium to large size with fine nuclear chromatin and nucleoli showing hypergranular cytoplasm. Occasional cells with 1 to 2 Auer rods (arrow) were seen but no faggot cell was detected. Right, Sudan Black B-stained positive blasts. Original magnification ×1000. (B) A karyotype performed on the diagnostic BM revealed 46,XX,t(5;11)(q31;p15). (C) Upper, a schematic diagram showing NUP98, JADE2, and NUP98-JADE2 generated by ProteinPaint. The phenylalanine-glycine (FG)/glycine-leucine-phenylalanine-glycine (GLFG) repeats and Gle2-binding-sequence (GLEBS) domain in the amino-terminal portion of NUP98 are retained in the fusion protein. Lower, RT-PCR analysis of NUP98-JADE2 (NJ) and JADE2-NUP98 (JN) fusions in the diagnostic BM. Whole BM cells were used for the analysis. Amplification of GAPDH served as the control. Expected products are indicated by arrowheads. Sanger sequencing of the NJ PCR product is also shown. (D) Immunofluorescence analysis of NUP98-JADE2 and wild-type JADE2. HeLa cells were transfected with pCMV-HA-NUP98-JADE2 or pCMV-HA-JADE2, and the tagged proteins were detected as described in supplemental Methods. DAPI was used for nuclear staining. Original magnification ×1000. (E) Interaction between NUP98-JADE2 and wild-type JADE2. 293T cells were transfected with the indicated Myc- and HA-tagged expression vectors. Myc-tagged proteins were immunoprecipitated and samples were analyzed by immunoblotting for NUP98-JADE2 (NJ) and JADE2 (J2) detection. Similar results were obtained when HA-tagged proteins were immunoprecipitated before immunoblotting (data not shown). (F) Altered subcellular distribution of wild-type JADE2 in the presence of NUP98-JADE2. Upper, HeLa cells were cotransfected with both pCMV-HA-NUP98-JADE2 and pCMV-Myc-JADE2, and the tagged proteins were detected as mentioned previously. Original magnification ×1000. Lower, HeLa cells were transfected with the indicated Myc- and HA-tagged expression vectors. Protein lysates were analyzed by immunoblotting. No apparent change in JADE2 expression was noted when NUP98-JADE2 was coexpressed.

Figure 2.

Molecular characterization of NUP98-JADE2 and JADE2 in ATRA-mediated responses and myeloid differentiation. (A) HeLa cells were cotransfected with RARE reporter and the indicated pCMV-HA plasmids. EV, empty vector; NJ, NUP98-JADE2; PR, PML-RARA. Luciferase activities were determined as described in supplemental Methods. (B) HeLa cells were cotransfected with RARE reporter and an increasing pCMV-HA-NUP98-JADE2 (NJ), pCMV-HA-JADE2 (J2), or the empty pCMV-HA (EV), and then treated with ATRA or DMSO for 6 hours before analysis. Results are presented as fold induction vs DMSO treatment. (A-B) *P < .05, **P < .01, and ***P < .001 vs EV, respectively by 1-way analysis of variance followed by Dunnett test. (C) NB4 cells transduced with NUP98-JADE2 (NJ) or control (EV) lentiviruses were treated with ATRA or DMSO. CD11b on GFP-positive cells was measured by flow cytometry. *P < .05 and **P < .01, respectively by paired t test. (D) Left, confirmation of JADE2 knockdown in NB4 cells after 24 hours of siRNA transfection by quantitative RT-PCR. After the transfection, cells were treated with 0.1 µM of ATRA or DMSO for 3 days. CD11b was measured by flow cytometry (middle) and CEBPE by quantitative RT-PCR and normalized to GAPDH (right). **P < .01 and ***P < .001 vs the negative siRNA, respectively by 1-way analysis of variance followed by Dunnett test. (E) Quantitative RT-PCR to validate selected JADE2 target genes in NB4 cells after 48 hours of siRNA transfection. **P < .01 vs negative siRNA by the Mann-Whitney U test. (A-E) Results are expressed as mean plus standard error from 3 independent experiments. (F) Pathway analysis of the 56 downregulated genes by ConsensusPathDB. The top 15 enriched Gene Ontology Biological Processes are shown. (G) Expression of the downregulated genes in purified polymorphonuclear cells (PMN) and granulocyte-monocyte progenitors (GMP). Data (from GSE42519) were available for 54 of the downregulated genes. Genes that are upregulated by ≥twofold (log₂ fc≥1) in PMN are boxed.