Chimeric NUP98-NSD1 transcripts from the cryptic t(5;11)(q35.2;p15.4) in adult de novo acute myeloid leukemia.
In the last years, collaborative studies have joined to link the degree of genetic heterogeneity of acute myeloid leukemia (AML) to clinical outcome, allowing risk stratification before therapy and guiding post-induction treatment of children with AML. So far, still half of these patients, whose disease is usually characterized by a grim prognosis, lack a known biomarker offering opportunities of targeted treatment. This relevant limitation prompted to pursue the search of new prognostic biomarkers to address also these forms of still uncharacterized AML. We chose to investigate NUP98 for its attitude to rearrange with different gene partners and produce oncogenic fusion transcripts often found to be associated with a wide range of hematological disorders, including leukemia. NUP98 rearrangements have been mainly mechanistically associated with oncogenic activation of HOX-AB cluster genes, a process largely documented for MLL-rearranged AML, in cooperation with other recurrent genetic defects, in particular FLT3-ITD. The frequency and prognostic relevance of NUP98 fusions in a single-center cohort of childhood AML has been recently reported, but neither the frequency nor the role of diverse NUP98 partners have been investigated. Here, we report an exhaustive molecular screening of 12 NUP98 rearrangements in the Italian pediatric patients with de novo AML enrolled in the AIEOP-AML 2002/01 trial. By RT-PCR, we characterized, at diagnosis, 494 patients harboring CBF rearrangements (RUNX1RUNX1T1 and CBFB-MYH11, N= 99), MLL-translocations (N= 86), FLT3-ITD (N= 54) and rarer mutations/translocations (N= 83), whereas in 172 patients no mutations were detected (35%). Among these 172 patients without known mutations and 36 patients with isolated FLT3-ITD mutation we searched for NUP98 rearrangements with NSD1, HOXC11, PHF23, HOXA9, JARID1A, HOXD13, LEDGF, DDX10, HHEX, ADD3, NSD3 and LOC348801, finding 16 (9.3%) with 6 different NUP98 fusions (16/172 = 9.3%) and 9 with t(5;11)NUP98-NSD1, respectively (9/36 = 25%, Supplementary Table 1S and 2S). In the 16 patients with NUP98 fusions, no CEBPA mutations were found, while 2 patients had K-RAS mutation, one of which concomitant with NPM1 mutation. Survival analyses revealed that the NUP98-t (N= 16) had a significant worse event-free survival (EFS, 25%) compared with the rest of AML (49.7%) patients without known mutations (N= 156, Neg in Figure 1a, Po0.01), and significantly higher incidence of relapse (Figure 1a, 66.3 vs 33.6% P-valueo0.01, Supplementary Table 1S). Reduced EFS and increased CIR were found also in the 9 NUP98-t patients with a concomitant FLT3-ITD mutation similar to isolated t(5;11) (Figure 1a). Collectively, in the whole AIEOP-AML trial cohort, we identified 25 NUP98-t patients (5%) characterized by a severe prognosis (Supplementary Figures 1SA–C). We then characterized NUP98-rearranged patients (n= 19) by using gene expression profiling (GEP) and compared them to 66 cases of AML with various genetic abnormalities (see Supplementary Methods, GSE75461). Supervised clustering showed that most of the NUP98-AML cases grouped independently of the rest of AML cases (Figure 1b, Fold Change4|1.5|, P-valueo0.01, Supplementary Figure 2SA, Supplementary Table 3SAB). In particular, the coding transcript clusters revealed 76 differentially expressed mRNAs (P-valueo0.05), where most of the upregulated genes were confirmed to belong to the HOX family, in particular the HOX-B cluster (see non-coding clustering analysis at Supplementary Figure 2SB), and the most downregulated were all genes involved in mitosis and nuclear division (CPNE8, CPNE3, CCNA1, FAS, DEFA423 being also validated by RQ-PCR Supplementary Figure 2SC). By gene ontology, we confirmed their involvement in the regulation of mitosis and chromatin modification (Supplementary Figure 2SD, Supplementary Table 4S), this finding supporting the role of NUP98 fusions in increasing genome instability. We further investigated this finding in ex vivo blasts obtained at diagnosis from t(5;11)NUP98-NSD1-rearranged patients and implanted in NSG mice (Supplementary Methods). We revealed spindle assembly checkpoint (SAC) defects in total cell extracts from NUP98-t cells arrested in M-phase after treatment with the microtubule-depolymerizing drug nocodazole (Noco), but no defects were found in BM cells from healthy donors (HBM) used as control. MAD2 and BUB1 protein levels decreased starting from 8 h post-Noco treatment, whereas Cyclin B levels increased, indicating that NUP98-t cells had an early and uncontrolled entrance in mitosis (Supplementary Figure 3SA). Then, we blocked DNA replication using aphidicolin (APH) and found an increased number of mitotic chromosomal breaks 24 h post treatment in NUP98-t cells compared with HBM cells (Supplementary Figure 3SB). This latter result was confirmed by protein analysis, which showed PP2A subunit β in the cytoplasm and phosphorylated H2AX (γH2AX) in the nucleus for the recruitment of enzymes to repair DNA in NUP98-t cells. This phenomenon was absent in HBM cells, where γH2AX was detected in the cytoplasm and PARP was found to be cleaved, suggesting that apoptosis occurred probably due to incapability of healthy cells to repair DNA and survive at the same APH dose (Figure 1c). We then moved to NUP98 partner gene characterization, finding evidence that NUP98-t patients significantly differentially expressed 101 transcript clusters independently of what was found in the rest of AML cases (60 coding and 41 non-coding RNAs, Supplementary Table 4S, Kruskal–Wallis test, P-valueo0.01; Figure 2a, Supplementary Table 5S). We focused our attention on HOX-AB cluster genes expression and found that, having NSD1 as partner gene, conferred the lowest HOX-AB expression among the six different NUP98-translocations (Figure 2a, Supplementary Figure 4SA, P-valueo0.05). Different GEP sustained a different survival depending on partner gene, with NSD1 mediating the worst prognosis (NSD1 N= 12; PHF23+JARID1A N=7, Supplementary Figure 4SBC, Po0.05). In order to identify peculiarities among the different NUP98 fusions, we performed enrichment analysis for several selected molecular signatures (Supplementary Methods). We found that all were enriched in chromosome instability and HOX/MLL signatures, with the exception of that involving PHF23, which showed correlation with methylation (Figure 2b, Supplementary Table 6S), and NUP98-JARID1A, confirmed to be typically involved in AML with megakaryoblastic features. This analysis recognized the NUP98-NSD1 rearranged patients being significantly enriched of the cAMP signaling, the GSK3 inhibitor pathway and CREB targets gene sets, identifying the cAMP response element binding protein (CREB) as a likely key player among these overrepresented signatures (Figure 2b, Fisher