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

Acute promyelocytic leukemia (APL) is featured by arrested promyelocytic differentiation and the PML-RARA fusion.¹  In variant APL, RARA is fused with other partners, which dictate disease biology and treatment sensitivity.²  Rarely, genomic rearrangements involving non-RARA genes have also been reported in patients with acute myeloid leukemia (AML) mimicking APL.^2-4  Although these findings suggest that alternative pathways may mediate the promyelocytic differentiation block, the underlying mechanisms have remained obscure. The nucleoporin 98 (NUP98) gene encoding a component of the nuclear pore complex is rearranged in various hematological malignancies associated with poor outcomes.^5,6  Here, we identify and characterize a novel NUP98-JADE2 fusion in a pediatric patient with AML with morphological and immunophenotypic features resembling APL. Our findings provided mechanistic insights into how a non-RARA rearrangement contributes to the development of an APL-like phenotype.

The patient was a 15-month-old girl who presented with fever and gum bleeding. Physical examination showed no pallor, jaundice, petechiae, bruises, or lymphadenopathy. Complete blood count revealed a hemoglobin level of 10.3 g/dL, platelet count of 227 × 10⁹/L, white blood cell count of 5.8 × 10⁹/L, and neutrophil count of 0.6 × 10⁹/L with 19% circulating blasts. Prothrombin time and activated partial thromboplastin time were 13.9 seconds (reference, 9.5-12 seconds) and 42.7 seconds (reference, 28.2-37.4 seconds), respectively. d-dimer levels were 4982 ng/mL (reference, <500 ng/mL). Bone marrow examination showed 61% of medium- to large-sized blasts with hypergranular cytoplasm (Figure 1A). The blasts were positive for CD13, CD15, CD33, CD117, and myeloperoxidase but negative for CD34, HLA-DR, CD14, CD41, glycophorin A, and lymphoid markers (CD10/CD19/CD20/CD22/CD79a/CD2/CD3/CD5/CD7/TdT) by flow cytometry. Nearly all the blasts showed Sudan Black B positivity. A diagnosis of APL was suspected and all-trans retinoic acid (ATRA; 25 mg/m² per day) was initiated while awaiting confirmatory results.

Reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization failed to detect PML-RARA or variant RARA fusions. Consistently, cytogenetic studies revealed 46,XX,t(5;11)(q31;p15)[20] (Figure 1B), indicating the absence of RARA translocations. No somatic mutation/copy number change in 141 myeloid-related genes (supplemental Table 1) including FLT3, NPM1, WT1, CEBPA, KIT, and K/NRAS was detected by targeted next-generation sequencing. In view of the PML-RARA negativity, ATRA was stopped after administration for 6 days and a shortened prothrombin time (12.9 seconds) and activated partial thromboplastin time (36.8 seconds) was noted. A diagnosis of AML not otherwise specified was made and the patient was treated with the Nordic Society of Paediatric Haematology and Oncology-AML 2004 protocol.⁷  Complete remission was achieved after the first induction course. However, the patient developed cardiac failure after the sixth course of high-dose cytarabine and etoposide and died 9 months after the initial diagnosis.

Detailed experimental procedures are provided in supplemental Methods. The study was approved by the institutional review board of the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Informed consent has been obtained from the parents of the patient.

RNA-sequencing revealed an in-frame NUP98-JADE2 fusion in which exon 13 of NUP98 was fused to exon 2 of JADE2, which encodes a plant homeodomain zinc finger protein (Figure 1C). The full-length fusion transcript is predicted to encode a 1304-amino acid protein containing the amino-terminal portion of NUP98 plus the entire JADE2. The reciprocal JADE2-NUP98 fusion was undetected. NUP98-JADE2 exhibited a speckled nuclear pattern in transfected HeLa cells (Figure 1D). On the contrary, wild-type JADE2 was uniformly and predominantly expressed in the nucleus. Coimmunoprecipitation studies in 293T cells showed that NUP98-JADE2 could heterodimerize with wild-type JADE2, suggesting that the fusion protein may perturb JADE2-associated pathways (Figure 1E). Accordingly, we found altered subcellular distribution of wild-type JADE2 with a speckled appearance overlapping with that of NUP98-JADE2 when the 2 proteins were overexpressed in HeLa cells (Figure 1F).

Deregulated RARA transcriptional control is central to APL pathogenesis.¹  The association of NUP98-JADE2 with APL-like features prompted us to examine the transcriptional properties of NUP98-JADE2 using a retinoic acid-response element (RARE)-luciferase reporter. Without ATRA, NUP98-JADE2 exerted no apparent effect on luciferase activity, whereas wild-type JADE2 mildly activated the reporter (Figure 2A). Intriguingly, NUP98-JADE2 dose-dependently inhibited ATRA-mediated transcriptional activation (Figure 2B; supplemental Figure 1). In contrast, no significant effect on luciferase induction was noted when wild-type JADE2 was overexpressed (Figure 2B). Consistent with these findings, overexpression of NUP98-JADE2 in NB4 and U937 cells diminished ATRA-induced CD11b expression, suggesting impaired myeloid differentiation (Figure 2C; supplemental Figure 2). Because NUP98-JADE2 altered wild-type JADE2 distribution and thus potentially affected its functions, we investigated the role of the wild-type protein. Interestingly, knockdown of JADE2 by small-interfering RNAs (siRNAs) in NB4 promyelocytic leukemia cells could similarly attenuate ATRA-induced expression of CD11b and CCAAT/enhancer binding protein ε (CEBPE) (Figure 2D), a marker of terminal granulopoiesis.⁸  Further transcriptome analysis of JADE2 knocked-down NB4 cells identified 89 genes whose expression was consistently altered (56 downregulated/33 upregulated) by ≥1.3-fold with 2 different siRNAs in 2 independent experiments (Figure 2E; supplemental Table 2). Pathway analysis revealed that the downregulated genes were enriched in inflammatory response, myeloid activation, and cytokine production (Figure 2F). In contrast, no enriched pathway (adjusted P < .05) was identified from the upregulated genes. By analyzing the expression profiles of the downregulated genes in purified myeloid cells, 65% of the genes were found to exhibit increased expression (≥twofold) during granulocytic maturation (Figure 2G). Knockdown of JADE2 also repressed RARE-mediated gene transcription (supplemental Figure 3).

In this study, we characterized a NUP98-JADE2 fusion in a pediatric patient with AML resembling APL and uncovered a role of JADE2 in myeloid differentiation. JADE2 is located at chromosome 5q31, a region frequently deleted in AML and myelodysplastic syndromes. The encoded protein has been shown to be a component of the histone acetyltransferase HBO1 complexes and a regulator of the histone demethylase LSD1/KDM1A.^9,10 NUP98-JADE2 is similar to other NUP98 fusions in multiple aspects including a preserved NUP98 amino-terminal portion, speckled nuclear distribution, aberrant HOXA and CDK6 expression (supplemental Figure 4), a histone recognition domain (the plant homeodomain) in the partner, and few cooperating mutations.^5,11,12  Concerning the latter, common AML-associated changes were absent in our case, implying that the fusion protein may be sufficient for leukemic transformation. On the other hand, NUP98-JADE2 is distinct from other NUP98 fusions in its association with an APL-like phenotype that is not observed with other non-RAR partners. However, the therapeutic efficacy of ATRA against NUP98-JADE2 is uncertain.

Localization to the correct cellular compartment is critical for proper functioning of a protein. The NUP98-JADE2-induced alteration of the subcellular distribution of JADE2 suggests that the fusion protein may act as a dominant-negative mutant to interfere with the wild-type protein. Concordantly, we observed comparable inhibitory effects of NUP98-JADE2 overexpression and JADE2 knockdown on ATRA-induced responses. On the other hand, how JADE2 perturbation affects ATRA-mediated transcriptional control remains incompletely understood. Because JADE2 is apparently a histone modulator, it is possible that the altered transcriptional response may involve epigenetic deregulation. Interestingly, JADE2 has been shown to promote degradation of LSD1,¹⁰  which has been implicated as a negative modulator of ATRA response.¹³  Thus, NUP98-JADE2 may impede ATRA response through LSD1 stabilization. A previous study has reported a presumed NUP98-JADE2 fusion in a juvenile myelomonocytic leukemia patient.¹⁴  However, the fusion breakpoints in both NUP98 and JADE2 were different and with concurrent NRAS/EZH2/KAT6A mutations, which were absent in our case.

In conclusion, we have identified a NUP98-JADE2 fusion conferring an APL-like phenotype in an AML patient and revealed a link between impaired retinoic acid signaling and the observed phenotype. Further studies characterizing the leukemogenic properties of NUP98-JADE2 shall shed new lights on APL pathogenesis.

The authors thank the Core Utilities of Cancer Genomics and Pathobiology (The Chinese University of Hong Kong) for providing the facilities and assistance in support of this research.

The work was partially supported by a grant from the General Research Fund program sponsored by the Research Grants Council in Hong Kong (CUHK M14108719).

Contribution: C.-K.C. designed and performed research and wrote the manuscript; H.-Y.C., Y.-L.Y., and T.S.K.W. performed research and analyzed data; A.W.K.L. and C.-K.L. managed the patient, collected clinical data, and advised on revision of the manuscript; K.T., N.P.H.C., and J.S.C. collected clinical samples and analyzed clinicopathological data; and M.H.L.N. designed and coordinated research and advised on revision of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Margaret H. L. Ng, Blood Cancer Cytogenetics and Genomics Laboratory, Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China; e-mail: margaretng@cuhk.edu.hk.