Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia

Abstract

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Figure 1.

Clinical characteristics of patients with and without NUP98 translocations. (A) Oncoprint depicting the major drivers of pediatric acute myeloid leukemia (AML) patients. (B) Circos plot depicting commonly co-occurring mutations and cytogenetic abnormalities in NUP98-translocated pediatric AML patients. (C) Pie charts depicting the sex divisions of patients in the NUP98-translocated AML subgroups. (D) Circos plot representing different fusion partner genes of NUP98-X translocations in pediatric AML patients. (E) Age distribution of AML patients. (F) Barchart (polar axis) illustrating the prevalence of NUP98 exon junctions in NUP98-translocated AML. The Figure legend is ordered by decreasing NUP98 exon prevalence in the NUP98 fusion-positive cohort.

Figure 2.

Cytogenetics of A/l/P9S-translocated pediatric acute myeloid leukemia. (A) Locations of breakpoints across the NUP98 gene for all NUP98-translocated acute myeloid leukemia (AML). (B) Oncoprint depicting additional copy number variations (CNV) and mutations in NUP98-translocated patients. (C) Heatmap depicting the presence and absence of flow-cytometry immunophenotype markers in NUP98-translocated AML groups. NUP98-HOX-like fusions include fusion partners HOXA9, HOXA13, HOXD13, and PRRX1. NUP98-Reader-like fusions include fusion partners BPTF, BRWD3, DDX10, HMGB3, KAT7, PHF15, SET, and TOP1.

Figure 3.

Expression patern of pediatric acute myeloid leukemia with various NUP98 translocations. (A) Unsupervised hierarchical clustering by gene expression including heterogenous pediatric acute myeloid leukemia (AML) subtypes, NUP98-translocated subgroups, normal healthy bone marrows (NBM) and CD34⁺ peripheral blood cells (CD34 PB). Annotation bars show AML subtype and co-occurring mutations. (B) Uniform manifold approximation and projection (UMAP) of gene expression, followed by Leiden clustering, for NUP98-translocated pediatric AML samples identifies five different transcriptional clusters. NUP98 fusions are indicated in different colors: NUP98-KDM5A in purple, NUP98-NSD1 in blue, and NUP98-X in green. (C) Expression of MECOM and PRDM16 genes in different subgroups of NUP98-translocated pediatric leukemia. Same identification colors for NUP98 fusions as in (B) are used. (D) Expression of stemness marker genes in all NUP98-translocated samples. Top bars represent French-American-British (FAB) classification and age category.

Figure 4.

Differential expression of all NUP98-translocated pediatric acute myeloid leukemia patients. (A) Schematic of differential expression analyses completed for NUP98-translocated samples. The overlap of differentially expressed genes (DEG) identified in each NUP98 cohort is represented in the Venn diagram. (B) DEG between NUP98-translocated AML groups compared to the reference cohort and normal bone marrow (NBM) were identified. Subsets of dysregulated genes were commonly identified in both NUP98-X and NUP98-NSD1 (upper panel) or were identified as shared between NUP98-X and NUP98-KDM5A (lower panel). (C) Commonly DEG found in all three NUP98-translocated pediatric acute myeloid leukemia (AML) subgroups. (D) Mean expression (Z-score transformed) of HOXA9 and HOXB8 interacting partners in NUP98-X translocated AML. The darker shades of red indicate higher expression in the NUP98-X cohort. CPM: counts per million.

Figure 5.

DNA methylation of pediatric acute myeloid leukemia patients with NUP98 translocations. (A) Uniform manifold approximation and projection (UMAP) of DNA methylation data in A/L^PSS-transLocated acute myeloid leukemia (AML) subgroups compared to the reference cohort and normal bone marrow (NBM). (B) Heatmap of non-negative matrix factorizations (NMF) of DNA methylation data. The NMF factors are those that were significantly associated with NUP98 translocation AML subgroups. (C) NMF factor associations of DNA methylation with NUP98-HOX-[\ke fusions (NUP98-NSD1, NUP98-HOX, and NUP98-PRRX1) and NUP98-Reader-like fusions (NUP98-KDM5A, NUP98- BPTF, NUP98-BRWD3, NUP98-DDX10, NUP98-KAT7, NUP98- PHF15, NUP98-SET, and NUP98-TOP1) with or without a co-occuring abnormal chromosome 3 (chr3). (D) NMF factor enrichments of DNA methylation for chromatin states, chromatin marks, and transcription factor binding sites. Factor 3 is enriched in NUP98-HOX-Like fusions, factor 6 is enriched in NUP98-Reader-like fusions, and factor 8 is enriched in NUP98-Reader-like fusions with an abnormal chr13. FDR: false discovery rate; OR: odds ratio; ReprPC: repressed PolyComb; TssAFlank: flanking active TSS, BivFlank: flanking bivalent; TSS/Enh, EnhBiv: bivalent enhancer; TxWk: weak transcription; Tx: Strong transcription; Enh: enhancers; EnhG: genie enhancers.

Figure 6.

Survival of pediatric acute myeloid leukemia patients with NUP98 translocations. Kaplan Meier estimates of (A) overall survival (OS) and (B) relapse risk (RR) of pediatric NUP98-translocated acute myeloid leukemia (AML) patients with different translocation partners compared to a reference cohort without NUP98 fusions. OS of (C) NUP98-NSD1 and (D) NUP98-X, when divided by NUP98 fusion exon breakpoint. Outcome was also examined for (E) OS and (F) event-free survival (EFS) of NUP98-KDM5A subgroups by chromosome 13 (chr13) status (monosomy 13, del(13q), translocation 13). Abn3: abnormal chr 3.