NUP98 Fusion Research Articles

Transcriptome-based molecular subtypes and differentiation hierarchies improve the classification framework of acute myeloid leukemia

The current classification of acute myeloid leukemia (AML) relies largely on genomic alterations. Robust identification of clinically and biologically relevant molecular subtypes from nongenomic high-throughput sequencing data remains challenging. We established the largest multicenter AML cohort (n = 655) in China, with all patients subjected to RNA sequencing (RNA-Seq) and 619 (94.5%) to targeted or whole-exome sequencing (TES/WES). Based on an enhanced consensus clustering, eight stable gene expression subgroups (G1-G8) with unique clinical and biological significance were identified, including two unreported (G5 and G8) and three redefined ones (G4, G6, and G7). Apart from four well-known low-risk subgroups including PML::RARA (G1), CBFB::MYH11 (G2), RUNX1::RUNX1T1 (G3), biallelic CEBPA mutations or -like (G4), four meta-subgroups with poor outcomes were recognized. The G5 (myelodysplasia-related/-like) subgroup enriched clinical, cytogenetic and genetic features mimicking secondary AML, and hotspot mutations of IKZF1 (p.N159S) (n = 7). In contrast, most NPM1 mutations and KMT2A and NUP98 fusions clustered into G6-G8, showing high expression of HOXA/B genes and diverse differentiation stages, from hematopoietic stem/progenitor cell down to monocyte, namely HOX-primitive (G7), HOX-mixed (G8), and HOX-committed (G6). Through constructing prediction models, the eight gene expression subgroups could be reproduced in the Cancer Genome Atlas (TCGA) and Beat AML cohorts. Each subgroup was associated with distinct prognosis and drug sensitivities, supporting the clinical applicability of this transcriptome-based classification of AML. These molecular subgroups illuminate the complex molecular network of AML, which may promote systematic studies of disease pathogenesis and foster the screening of targeted agents based on omics.

FLT3ITD drives context-specific changes in cell identity and variable interferon dependence during AML initiation

AbstractAcute myeloid leukemia (AML) initiation requires multiple rate-limiting mutations to cooperatively reprogram progenitor cell identity. For example, FLT3 internal tandem duplication (FLT3ITD) mutations cooperate with a variety of different initiating mutations to reprogram myeloid progenitor fate. These initiating mutations often skew toward either pediatric or adult AML patient populations, though FLT3ITD itself occurs at similar frequencies in both age groups. This raises the question of whether FLT3ITD might induce distinct transcriptional programs and unmask distinct therapeutic vulnerabilities when paired with pediatric, as opposed to adult AML-initiating mutations. To explore this possibility, we compared AML evolution in mice that carried Flt3ITD/NUP98-HOXD13 (NHD13) or Flt3ITD/Runx1DEL mutation pairs, which are respectively most common in pediatric and adult AML. Single-cell analyses and epigenome profiling revealed distinct interactions between Flt3ITD and its cooperating mutations. Whereas Flt3ITD and Flt3ITD/Runx1DEL caused aberrant expansion of myeloid progenitors, Flt3ITD/NHD13 drove the emergence of a pre-AML population that did not resemble normal hematopoietic progenitors. Differences between Flt3ITD/Runx1DEL and Flt3ITD/NHD13 cooperative target gene expression extended to fully transformed AML as well. Flt3ITD/NHD13 cooperative target genes were enriched in human NUP98-translocated AML. Flt3ITD/NHD13 selectively hijacked type I interferon signaling to drive expansion of the pre-AML population. Blocking interferon signaling delayed AML initiation and extended survival. Thus, common AML driver mutations, such as FLT3ITD, can coopt different mechanisms of transformation in different genetic contexts. Furthermore, pediatric-biased NUP98 fusions convey actionable interferon dependence.

Tracking Clonal Evolution in Pediatric AML

Acute myeloid leukemia (AML) makes up nearly 20% of acute pediatric leukemias and has a long-term survival rate of approximately 70%. A key driver of the low overall survival for children with AML is relapsed disease and less than 40% of the children with relapsed AML will survive past five years. We recently characterized relapsed pediatric AML (pAML) to better understand the spectrum of genomic alterations at disease recurrence (Umeda et al., 2022). However, the genome-wide patterns of genetic evolution from diagnosis to relapse have yet to be characterized for pAML. Thus, there is a gap in knowledge of how alterations at diagnosis evolve during treatment to drive relapse and poor outcome. We performed whole genome sequencing (WGS) and whole transcriptome sequencing (RNA-Seq) on a cohort of 50 pediatric AML patients with paired diagnosis and relapse samples. The median age at diagnosis was 10.7 yrs (range 0.7-20yrs), and the median time between diagnosis and relapse was 334 days (range 93-1,773 days). The main driver group contained a KMT2A fusion event (n=16, 32%). NUP98 fusion made up the second largest group at 14% (n=7). Also within the cohort are newly defined categories, including a BCL11B structural variant (n=1) and patients with UBTF tandem duplication AML (n=4). These features are overall similar to the molecular profiles of pediatric AML (Farrar et al., 2016; Umeda et al., 2022). Approximately 22,500 alterations (including point mutations, indels, and structural variants) were detected by WGS (median coverage 36X). The median number of alterations at diagnosis and relapse per patient was, respectively, 224 (range 118-867) and 411 (range 73-1,909). Most of the cohort noticed more alterations at relapse than diagnosis, but some (n=8) had fewer at relapse. Additionally, there was no trend with the time to relapse and the number of acquired alterations. The most commonly mutated genes regardless of timepoint included FLT3 (n=16, 6 were internal tandem duplications), WT1 (n=18), NRAS (n=16), and SETD2 (n=9). We identified over 11,000 relapse enriched variants (median 169, range 3-1,378), including alterations that had a significantly higher allele frequency (q-value < 0.05) at relapse compared to diagnosis. This included 1,028 relapse enriched variants that alter protein coding genes. FLT3 contained 7, 2, and 7 relapse enriched, relapse depleted, and time point stable alterations, respectively. WT1 contained 6, 2, and 10 relapse enriched, relapse depleted, and time point stable alterations, respectively. Furthermore, the relapse-enriched list included three SETD2 protein-truncating mutations and four AUTS2 structural variants often co-occurring with KMT2A fusions, indicating that these mutations may cooperate with KMT2A fusion and contribute to the disease progression. To increase our understanding of the clonal structure, 36 cases with additional DNA material were re-sequenced with a custom panel covering 6,300 variants, including all coding variants for each case. This led to a validation rate of 97% of all variants and the increased sequencing depth of the panel (median 265X for diagnosis and 274X for relapse) gave greater insight into the changes in clonal structure between diagnosis and relapse time points. Most of the cohort (70%) featured a relapsed enriched clone that was minor at diagnosis and became clonal in relapse. Additional cases (n=10) featured a clone that was undetectable by WGS at diagnosis and after treatment became the dominant clone in relapse. Only one case maintained the same major clone in both diagnosis and relapse. In summary, our data show a comprehensive characterization of changes in the clonal structure of pAML between diagnosis and relapse, revealing relapse-enriched gene alterations which may be driving relapse disease. Farrar, J. E., Schuback, H. L., Ries, R. E., Wai, D., Hampton, O. A., Trevino, L. R., . . . Meshinchi, S. (2016). Genomic Profiling of Pediatric Acute Myeloid Leukemia Reveals a Changing Mutational Landscape from Disease Diagnosis to Relapse. Cancer Res, 76(8), 2197-2205. doi:10.1158/0008-5472.CAN-15-1015 Umeda, M., Ma, J., Huang, B. J., Hagiwara, K., Westover, T., Abdelhamed, S., . . . Klco, J. M. (2022). Integrated genomic analysis identifies UBTF tandem duplications as a recurrent lesion in pediatric acute myeloid leukemia. Blood Cancer Discov. doi:10.1158/2643-3230.BCD-21-0160 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

105. Clinical whole-genome sequencing identifies NSD3 as the correct fusion partner of NUP98 in a patient with acute myeloid

<h3>Background</h3> Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. <h3>Case Presentation</h3> Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. <h3>Conclusions</h3> NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

AML-283 The Genetic Landscape of NUP98-Rearranged Pediatric Leukemia

Rearrangements involving NUP98 are frequently found in pediatric acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL) and are associated with poor prognosis. Various fusion partners have been identified; however, the genetic basis of how different fusion genes and cooperating mutations contribute to the disease phenotypes and the refractory nature remains to be clarified. To elucidate the transcriptional and genetic background of pediatric NUP98-rearranged acute leukemia. Transcriptomic and genetic analyses were performed on pediatric leukemia with NUP98 rearrangements from multiple clinical studies at St. Jude Children's Research Hospital, by the Italian Association for Pediatric Hematology and Oncology, or by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. No blinding was done in this study. RNA sequencing from 95 patients (median age of 9.0 years) with NUP98-rearranged (NUP98r) leukemia was performed or obtained from previous studies. Eighty-one of these samples were also studied by tumor-normal-paired whole-genome or whole-exome sequencing. We identified 17 unique NUP98 fusion partners from RNAseq data with NSD1 as the most frequent partner (n=44, 46.3%), followed by KDM5A (n=27, 28.4%) and RAP1GDS1 (n=6, 6.3%). We also found rare fusion partners recurrent in this cohort, including LNP1, HOXA13, and HOXD13 (n=2, 2.1%, respectively). Among cooperating mutations, FLT3-ITD and WT1 were frequent in NSD1-AML (57.9% and 52.6%, respectively), and copy number loss of chromosome 13 harboring RB1 was found in 86.3% of NUP98-KDM5A AML with erythromegakaryocytic expression patterns (FAB M6-7). NUP98-RAP1GDS1 fusions are found in both AML (n=3) and T-ALL (n=3) cases and those with T lineage differentiation have concurrent NOTCH1 mutations, indicating that both fusions and cooperating mutations are contributing to the disease phenotypes. We further performed transcriptome analysis of NUP98r AML, integrating various AML subtypes from previous studies. NUP98r AML expressed high MEIS1, PRDM16, and HOXA-B cluster genes similar to AMLs with NPM1 or UBTF mutations, suggesting a molecular basis shared by these subtypes. Genomic and transcriptional profiling of pediatric NUP98-rearranged leukemia identified unique associations among fusions, cooperating mutations, and disease phenotypes.

Abstract 3479: UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome

Abstract Childhood AML is an aggressive disease with high rates of failures and poor survival. We have demonstrated that the molecular landscape of AML in children is distinct, and co-occurrence of variants modulate outcomes. Recent discovery of tandem duplication (TD) of the UBTF gene in AML, with enrichment in FLT3-ITD has implicated yet another mutation whose cooperation with FLT3-ITD may modify outcome. Here, we provide a comprehensive evaluation of UBTF-TD in de novo AML and define its clinical implications within FLT3-ITD patients. Initial interrogation of transcriptome data from 1,158 children enrolled on COG AAML1031 identified 50 cases of UBTF-TD (4.3%). Overwhelming majority of UBTF-TD cases were observed in FLT3-ITD cases (77%), vs. that of 1.2% in those without FLT3-ITD (p&amp;lt;0.001). Given extreme enrichment of UBTF-TD in FLT3-ITD, we inquired whether cooperation of UBTF-TD and FLT3-ITD creates a distinct clinical entity. To this end we screened diagnostic DNA from 400 FLT3-ITD patients treated on three consecutive CCG/COG trials (COG AAML1031, COG AAML0531, and CCG-2961) by PCR and fragment analysis. UBTF-TD was identified in 61 FLT3-ITD cases (15.3%). The data presented here forth focuses on evaluation of implications of UBTF-TD in FLT3-ITD positive patients only. Within the FLT3-ITD patients, initial correlation of UBTF-TD with demographics, disease characteristics, and associated genomic variants was conducted. Patients with and without UBTF-TD had a similar median age at diagnosis (p=0.322), lower diagnostic WBC (p=0.010) and higher marrow blast % (p&amp;lt;0.001). There was a stark paucity of cooperating variants that commonly co-occur with FLT3-ITD, with a single NPM1 mutation (1.6% vs. 29%, p&amp;lt;0.001) and no NUP98 fusions (0% vs. 23%, p&amp;lt;0.001). There was a significant enrichment of WT1 mutations, with 45% UBTF-TD patients with a WT1 mutation (FLT3-ITD/UBTF-TD/WT1), vs. 11% in UBTF-WT (p&amp;lt;0.001). Trisomy 8 (Tri8) was seen in 15% of UBTF-TD. Patients with UBTF-TD had a lower CR rate (44% vs. 60%, p = 0.018), and Higher MRD rate (38% vs. 21%, p&amp;lt;0.001). Patients with and without UBTF-TD had an EFS of 28% vs. 42% (p=0.047) with a corresponding OS of 40% and 57% (p=0.019). Given enrichment of WT1 mutations and Tri8 in patients with UBTF-TD, we studied the outcome UBTF-TD patients in the context of these two variants. FLT3-ITD/UBTF-TD/WT1 patients had a 5-year EFS of 17% vs. 38% for similar patients without WT1 mutations (p=0.0062). Patients with UBTF-TD with additional Tri8 had a similarly poor outcome with an EFS of 23% with a corresponding OS of 33%, providing a distinct high risk UBTF-TD cohort (+WT1 or Tri8), whereas the remaining UBTF-TD patients had a more favorable outcome with EFS and OS of 64% and 86%, respectively (p&amp;lt;0.0001, and p&amp;lt;0.0001). UBTF-TD is a novel genomic entity with high enrichment in patients with FLT3-ITD and a distinct clinical outcome driven by cooperating WT1 mutation and Tri8. Citation Format: Leila Robinson, Amanda Leonti, Todd A. Alonzo, Yi-Cheng Wang, Michele S. Redell, Rhonda E. Ries, Jenny L. Smith, Tiffany A. Hylkema, Quy Le, E Anders Kolb, Richard Aplenc, Xiaotu Ma, Jeffrey Klco, Katherine Tarlock, Soheil Meshinchi. UBTF tandem duplications (UBTF-TD) in childhood AML: Enrichment in FLT3-ITD and association with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3479.

The role of phase separation in oncogenesis by fusion oncoproteins

NUP98 fusion oncoproteins (FOs) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid‐liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98‐HOXA9 (NHA9), retains a DNA‐binding homeodomain, whereas others harbor other types of DNA‐ or chromatin‐binding domains. NUP98 FOs have long been known to form puncta, but long‐standing questions are how nuclear puncta form, and how they drive leukemogenesis. We will discuss our studies of NHA9 condensates, showing that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three other leukemia‐associated NUP98 FOs form nuclear puncta and transform hematopoietic cells. To extend our findings, we tested ~150 additional fusion oncoproteins associate with a wide range of human cancers for puncta formation in cells. We will discuss our observation that &gt;50% of these formed cellular condensates, with this behavior for some linked with aberrant gene expression and cell transformation. Our findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs and likely contributes to oncogenesis driven by many other fusion oncoproteins.

In This Issue

In This Issue

Emerging Targeted Therapy for Specific Genomic Abnormalities in Acute Myeloid Leukemia.

We describe recent updates of existing molecular-targeting agents and emerging novel gene-specific strategies. FLT3 and IDH inhibitors are being tested in combination with conventional chemotherapy for both medically fit patients and patients who are ineligible for intensive therapy. FLT3 inhibitors combined with non-cytotoxic agents, such as BCL-2 inhibitors, have potential therapeutic applicability. The menin-MLL complex pathway is an emerging therapeutic target. The pathway accounts for the leukemogenesis in AML with MLL-rearrangement, NPM1 mutation, and NUP98 fusion genes. Potent menin-MLL inhibitors have demonstrated promising anti-leukemic effects in preclinical studies. The downstream signaling molecule SYK represents an additional target. However, the TP53 mutation continues to remain a challenge. While the p53 stabilizer APR-246 in combination with azacitidine failed to show superiority compared to azacitidine monotherapy in a phase 3 trial, next-generation p53 stabilizers are now under development. Among a number of non-canonical approaches to TP53-mutated AML, the anti-CD47 antibody magrolimab in combination with azacitidine showed promising results in a phase 1b trial. Further, the efficacy was somewhat better in patients with the TP53 mutation. Although clinical evidence has not been accumulated sufficiently, targeting activating KIT mutations and RAS pathway-related molecules can be a future therapeutic strategy.

The Retinoblastoma Tumor Suppressor Is Required for the NUP98-HOXA9-Induced Aberrant Nuclear Envelope Phenotype.

Chromosomal translocations involving the nucleoporin NUP98 gene are recurrently identified in leukemia; yet, the cellular defects accompanying NUP98 fusion proteins are poorly characterized. NUP98 fusions cause changes in nuclear and nuclear envelope (NE) organization, in particular, in the nuclear lamina and the lamina associated polypeptide 2α (LAP2α), a regulator of the tumor suppressor retinoblastoma protein (RB). We demonstrate that, for NUP98-HOXA9 (NHA9), the best-studied NUP98 fusion protein, its effect(s) on nuclear architecture largely depend(s) on RB. Morphological alterations caused by the expression of NHA9 are largely diminished in the absence of RB, both in human cells expressing the human papillomavirus 16 E7 protein and in mouse embryonic fibroblasts lacking RB. We further show that NHA9 expression associates with distinct histone modification. Moreover, the pattern of trimethylation of histone H3 lysine-27 is affected by NHA9, again in an RB-dependent manner. Our results pinpoint to an unexpected interplay between NUP98 fusion proteins and RB, which may contribute to leukemogenesis.

The menin-MLL1 interaction is a molecular dependency in NUP98-rearranged AML

AbstractTranslocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion-driven leukemia is sensitive to the menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of proleukemogenic transcription factors such as Meis1 in NUP98-fusion-transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for the maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion-driven AML to test the in vivo efficacy of menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that menin-MLL1 inhibition simultaneously suppresses a proleukemogenic gene expression program, including downregulation of the HOXa cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Profiling Defines Distinct Prognostic Subgroups in Chilhood Acute Myeloid Leukemia: A Report from the French ELAM02 Study Group

Introduction:Despite major treatment improvements over the past decades, pediatric acute myeloid leukemia (AML) is still a life-threatening malignancy with relapse rates up to 30% and survival rates below 75%. Moreover, most of investigations are based on the study of large cohorts of adult AML patients while genetic profiles are known to be quite different between adults and children with AML. In this context, a better description of the pattern of molecular aberrations in childhood AML remains a great challenge to refine prognostication and improve outcome in such patients. We report here the comprehensive molecular landscape of a large and well-annotated cohort of pediatric AML and propose a new prognostic molecular classifier in this particular group of patients.Patients and methods:We performed molecular analysis using both high-throughput sequencing focused on 36 genes recurrently mutated in myeloid malignancies and ligation-dependent RT-PCR detecting more than 50 recurrent gene rearrangements in 385 patients with AML of the 438 children enrolled in the prospective ELAM02 trial and we evaluated their prognostic significance.Results:The median age at AML diagnosis was 8.6 years (range: 0-18) and the median WBC count was 16.6×109/L (range: 0.40-575). The distribution in the cytogenetic subgroups was as follows: normal karyotype (n=101, 26.2%), CBF-rearranged (n=92, 24% including t(8;21): n=57 and inv(16)/t(16;16): n=35), KMT2A (MLL)-rearranged (n=79, 21%), adverse karyotype (n=40, 10% including complex karyotype: n=27, monosomy 7: n=9 and t(6;9): n=4) and other (n=73, 19%). 76% of patients had at least one driver mutation among the genes we screened. The most common class of mutations involved genes that control tyrosine kinase signaling (61% including CBL, FLT3, JAK2, KIT, KRAS, MPL, NRAS, PTPN11, SETPB1) followed by transcription factors (16%: CEBPA, ETV6, GATA1, GATA2, RUNX1), tumor suppressors (14%: PHF6, PTEN, TP53, WT1), NPM1 (9%), chromatin modifiers (9%: ASXL1, BCOR, BCORL1, EZH2), DNA methylation controllers (8%: DNMT3A, IDH1, IDH2, TET2), cohesin genes (5%: NIPBL, RAD21, SMC1A, SMC3, STAG2) and spliceosome (3%: SF3B1, SRSF2, U2AF1, ZRSR2). Moreover, a recurrent transcript fusion was detected in about a half of pediatric patients. Taken together, we identified at least one molecular aberration (mutations or fusion transcripts) in 344 (89%) out of 385 patients.In multivariate analysis, the presence of a NUP98 fusion is the sole variable associated with induction failure (50% CR, P=0.038). NUP98 -rearranged cases represented 2.6% of this cohort (10 patients/385 in which 5 had a normal karyotype). When studying OS, 5 factors revealed a negative impact : (1) WBC count higher than 30×109/L (P=0.001); (2) WT1 mutations (P=0.027); (3) RUNX1 mutations (P=0.043); (4) PHF6 mutations (P=0.038) and (5) adverse cytogenetics (P<0.001). On the other hand, 3 factors were shown to positively impact OS: (1) NPM1 mutations (P<0.001), (2) CEBPA dm (P=0.042) and (3) CBF-rearrangements (P<0.001).Considering results from multivariate analysis and strong molecular markers validated among studies, we defined a molecular classifier, refining the prognosis in childhood AML (see figure). Overall, CBF rearrangements, NPM1 and double CEBPA mutations represented 37% of the cohort and defined a favorable molecular subgroup (3-years OS: 92%) while NUP98 fusions, WT1, RUNX1 and PHF6 mutations (15% of the cohort) segregated into a poor molecular subgroup (3-years OS: 46%). Neither karyotype nor others gene mutations were able to discriminate patients into the intermediate molecular risk subgroup (3-years OS: 73%). KMT2A -rearrangements (21% of the cohort) were associated with an intermediate risk.Conclusion:Despite some overlaps, the spectrum of molecular aberrations and their prognostic significance differ between childhood and adult AML. These results have important implications to contribute in refining risk stratification of pediatric AML and show the need for further validations in independent pediatric cohorts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

Acute erythroid leukemia is enriched in NUP98 fusions: a report from the Children's Oncology Group

AbstractAcute erythroid leukemia (AEL) is a rare subtype of acute myeloid leukemia (AML) primarily affecting older adults and was previously classified into erythroid/myeloid and pure erythroid subtypes. In this pediatric AEL study, we evaluated morphologic, immunophenotypic, cytogenetic, molecular, and clinical data of 24 (1.2%) cases from all cases undergoing central pathology review in Children's Oncology Group trials AAML0531 and AAML1031. Of 24 cases, 5 had a pure erythroid phenotype, and 19 had an erythroid/myeloid phenotype. NUP98 fusions were highly enriched in patients with AEL, occurring in 7 of 22 cases for which molecular data were available (31.8% vs 6.7% in other AML subtypes). Of 5 cases of pure erythroid leukemias (PELs), 3 had NUP98 fusions, and 4 had complex karyotypes. Erythroid/myeloid leukemias were reclassified by using the 2017 World Health Organization hematopathology classification as: myelodysplastic syndrome (MDS) with excess blasts-1 (n = 3), MDS with excess blasts-2 (n = 7), AML (nonerythroid, n = 5), and unknown MDS/AML (n = 4); the 5 cases of nonerythroid AML included 1 with an NUP98–NSD1 fusion, 2 with myelodysplasia-related changes, and 1 with a complex karyotype. Three cases of MDS with excess blasts-2 also had NUP98 rearrangements. WT1 mutations were present in 5 of 14 cases, all erythroid/myeloid leukemia. Outcomes assessment revealed statistically poorer overall survival (5-year, 20% ± 36% vs 66% ± 23%; P = .004) and event-free survival (5-year, 20% ± 36% vs 46% ± 23%; P = .019) for those with PEL than those with erythroid/myeloid leukemia. Our study supports that AEL is a morphologically and genetically heterogeneous entity that is enriched in NUP98 fusions, with the pure erythroid subtype associated with particularly adverse outcomes.

The Molecular Characteristics and Clinical Relevance of NUP98-Other Translocations in Pediatric Acute Myeloid Leukemia

Background Cytogenetic and molecular aberrations are important prognostic factors in pediatric acute myeloid leukemia (AML). NUP98 translocations with more than 30 different partner genes have been identified in pediatric AML. The 2 most common fusions, NUP98-NSD1 and NUP98-KDM5A, have been shown to have distinct characteristics and are both associated with adverse outcomes. Although NUP98 fusions with less common fusion partners have been identified, the biological and clinical implications of these variants are unknown. Methods To determine the biological and clinical implications of the less common "other" NUP98 translocations (NUP98-X), we evaluated the clinical characteristics and transcriptome and genome sequencing data from 2396 children and young adults with AML within 4 consecutive Children's Cancer Group (CCG) and Children's Oncology Group (COG) trials CCG-2961, AAML03P1, AAML0531 and AAML1031. All NUP98-X translocations were confirmed by RNA sequencing. Results Of the 2396 patients screened, 164 patients (6.8%) had a NUP98 translocation. We identified 20 patients with a NUP98-X fusion (0.83%) and compared them with those with NUP98-NSD1 (n=110, 4.5%), NUP98-KDM5A(n=34, 1.4%), and a reference cohort without NUP98 translocations (n=2232). Translocation partners identified in the NUP98-X group were HOXA9 (n=4), HOXD13 (n=3), PHF15 (n=2), PHF23 (n=2), and single cases of BPTF, BRWD3, DDX10, HMGB3, HOXA13, KAT7, PRRX1, SET and TOP1. Besides the distinct characteristics of NUP98-NSD1 and NUP98-KDM5A, the NUP98-X group showed high inter-patient variance in clinical characteristics compared to our reference cohort. NUP98-X patients showed a clear bimodal age distribution with half of the patients being in the older age category and a similar number in the category &amp;lt;3 years (Figure 1). White blood cell count and blast percentages were in line with those of the reference cohort. We investigated the co-occurrence of common pediatric AML mutations within our cohort (Figure 2). There was a lack of additional driving mutations with the exception of WT1 mutations that are significantly more prevalent in the NUP98-X group compared to the reference cohort (25% vs 8%, p=0.018). NUP98-X patients with a WT1 mutation have a median age of 16.3 years compared to 2.3 years for those without a WT1 mutation. Also, in contrast to NUP98-NSD1 translocated patients, the co-occurrence of FLT3-ITD did not occur in NUP98-X translocated patients. Further, NUP98-X patients had a more varied transcriptome profile than did patients with NSD1 or KDM5A translocations (Smith J, et al. ASH abstract 2020). We evaluated the impact of NUP98-X translocations in response to the initial induction therapy. The morphological complete remission (CR) rate after course 1 was 65% in the NUP98-X cohort versus 76% in the reference cohort (p=0.266). NUP98-NSD1 patients had an inferior CR rate (36%, p&amp;lt;0.001), but the CR rate in NUP98-KDM5A patients was similar (77%) to the reference cohort. Of all NUP98-X patients, 11% underwent stem cell transplantation (SCT), compared to 17% in the reference cohort, 40% in the NUP98-NSD1 and 23% in the NUP98-KDM5A cohort. Outcome analysis for NUP98-X patients demonstrated that despite differences in disease characteristics, they have similar adverse outcomes as patients with NSD1 or KDM5A translocations (Figure 3). Overall survival (OS) at 5 years from diagnosis was 35% compared to 65% in the group without NUP98 translocations (p=0.006, Figure 3). Event-free survival was 35% for NUP98-X patients, compared to 48% for those without NUP98 translocations (p=0.282). These adverse outcome rates are comparable to those reported in studies on high-risk pediatric AML patients. Conclusion NUP98-X translocated pediatric AML patients represent a rare cohort with a high variability in both translocation partners and other clinical characteristics. Despite this heterogeneity, the OS of these patients is comparable to high-risk pediatric AML patients, which justifies a high-risk stratification of these patients and emphasizes the need for developing new treatment strategies. Further research within large patient cohorts is needed to investigate the biologic and clinical characteristics of these rare translocations and potential differences between the different NUP98-X fusion partners. Disclosures Kaspers: AbbVie: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&amp;D: Ended employment in the past 24 months. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019).

The Transcriptomic Landscape of NUP98-Rearranged Pediatric Acute Myeloid Leukemia

Childhood acute myeloid leukemia (AML) is an aggressive myeloid malignancy characterized by mutational and cytogenetic abnormalities. Chromosomal rearrangements involving the NUP98 gene have come to light for its' significant impacts on outcome and response to treatment. NUP98-rearranged (NUP98-R) AML includes NUP98-NSD1, NUP98-KDM5A, and various less common NUP98 fusion partners - such as HOX, SET, and Bromodomain genes. NUP98-R account for 6.8% of patients across Children's Oncology Group studies CCG2961, AAML03P1, AAML0531, and AAML1031. The majority are NUP98-NSD1 (4.6%), then NUP98-KDM5A (1.4%), and the various partners, termed NUP98-X (0.83%). However, the biological implications of NUP98-X have yet to be investigated. We define transcriptional clusters and report transcriptional profiling results to reveal similarities and differences between the diverse NUP98-R fusions. RNA sequencing was completed for 1,492 pediatric AML patients and 68 healthy bone marrow controls (NBM). The algorithms STARfusion, TransAbyss, and CICERO detected 156 NUP98-R from RNA seq: NUP98-NSD1 (N=104), NUP98-KDM5A (N=32), and NUP98-X (N=20). NUP98-X encompassed 13 unique fusion partners and 45% (9/20) had a homeobox gene partner - HOXA9 (N=4), HOXD13 (N=3), HOXA13 (N=1), and PRRX1 (N=1). Unsupervised clustering via uniform manifold approximation and projection (UMAP) with input genes selected by jackstraw PCA revealed NUP98-X cluster more closely with NUP98-NSD1, but their trajectory is dispersed within NUP98-NSD1 and NUP98-KDM5A, indicating a potential hybrid transcriptional profile (Fig 1 A). NUP98-NSD1 clustered remotely from the majority of NUP98-KDM5A, highlighting their unique transcriptional profiles despite sharing NUP98-R. Five transcriptional clusters were identified by the Leiden algorithm, followed by selection of genes significantly associated with cluster assignment. Cluster marker genes were filtered to be unique for each cluster prior gene-set enrichment analysis (Fig 1 B). Cluster C3 encompassed the largest proportion of NUP98-X, with 7 homeobox and both PHF23 cases (9/20, 45%), but also comprised of 19 NUP98-NSD1 and 2 NUP98-KDM5A. Importantly, C3 was highly associated with the expression of long non-coding RNAs, Z69666.2, MT1XP1, and RP11-455O6.2 (≥ 37% specificity, FDR &amp;lt; 0.001), and pathways in enriched in oncogenic microRNAs (FDR &amp;lt; 0.001), suggesting a non-coding signature may define this group. NUP98-NSD1 divided into two primary clusters, C1 (52/104) and C2 (31/104), differentiated by the expression of mannose receptor genes MCR1/ MRC1L1, which detect products of the lysosome pathway, significantly activated in C1 (FDR &amp;lt; 0.001). NUP98-KDM5A also segregated into two clusters, C4 (N=22/32) and C5 (N=7/32) based on an FAB M7/AMKL signature, with 78.6% of M7 NUP98-R located in C4 (p=0.003). Whereas, C5 cases were non-M7 and uniquely enriched in the regulation of stem cells pathway (FDR = 0.005); activation was driven by the expression of POU5F1, BMP4, WNT7, and WNT10A/B. Transcriptional profiling of NUP98-R cohorts, independently compared to other AML (N=1,336), found 27 upregulated differentially expressed genes (DEGs) shared between NUP98-X and NUP98-NSD1, including MYCN oncogene, homeobox PBX3, and DNA methyltransferase DNMT3B; on the other hand, shared overexpression of MLLT3, homeobox IRX3, and CD79a characterized similarities between NUP98-X and NUP98-KDM5A (Fig 1C) and may contribute to the hybrid expression profile observed by UMAP clustering. There were 38 DEGs shared between all NUP98-R cohorts. Differential expression analysis comparing NUP98-R cohorts vs NBM (N=68) showed that 22/38 shared DEGs were dysregulated in normal hematopoiesis and 15/38 genes were concordantly overexpressed in NUP98-R. The minimal set of 15 genes strongly implicates dysregulation at the HOX locus; these targets include hsa-mir-10a, whose genomic locus is within the HOXB cluster, CACNG4 located on chr17q, the same chromosome arm as HOXB, and the remaining shared targets were HOXA/B genes (Fig 1D). In summary, we found that NUP98-X are dispersed between NUP98-NSD1 and NUP98-KDM5A by transcriptional clustering. Clustering revealed significant pathways and marker genes that may contribute to segregation of NUP98-R cohorts, and DEGs that contribute in part to their hybrid transcriptional profile, including HOX genes, PBX3, and IRX3. Disclosures No relevant conflicts of interest to declare.

Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the β-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

Abstract B34: Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions

Abstract Chromosomal translocations of the Nucleoporin 98 gene (NUP98) are associated with multiple hematopoietic malignancies. In particular, NUP98 rearrangements can be found in up to 6.6% of de novo cases of pediatric AML. Specific subpopulations of pediatric AML appear to be even more enriched for NUP98 fusions, with over 16% of cytogenetically normal pediatric AML patients and 15% of non-Down syndrome acute megakaryoblastic leukemia (AMKL) patients harboring these fusions. Clinically, the NUP98-KDM5A fusion is associated with erythroid and megakaryocytic leukemias while the NUP98-NSD1 fusion is associated with myelomonocytic leukemias. When NUP98 fusions are present in AML, they confer a poor prognosis. To better characterize the role of these fusions in human hematopoiesis, we used cord blood CD34+ (cb-CD34+) hematopoietic stem and progenitor cells (HSPCs) as an in vitro model system. We transduced cb-CD34+ HSPCs with NUP98-NSD1, NUP98-KDM5A, or empty vector control and studied the effects on cell proliferation and differentiation in liquid culture media containing cytokines (IL-6, FLT3-Ligand, SCF, TPO, and SR1 (an aryl hydrocarbon receptor antagonist)). NUP98-NSD1 and NUP98-KDM5A fusions increase the proliferation rate in liquid culture by three-fold and 19-fold, respectively (p&amp;lt;0.05). Consistent with observations from patients with these fusions, cells expressing NUP98-KDM5A have increased expression of CD235A and CD71, consistent with erythroid differentiation (p&amp;lt;0.05). In addition, cells expressing NUP98-NSD1 have increased expression of CD11b compared to NUP98-KDM5A (p&amp;lt;0.01) expressing cells. When placed in methylcellulose, the NUP98 fusions tested had no effect on total colony number, but we observed more erythroid differentiation for NUP98-KDM5A compared to vector control (p&amp;lt;0.05). Overall, the results of these in vitro studies demonstrate increased proliferation and inhibition of differentiation in HSPCs by NUP98 fusions, and cell differentiation patterns parallel the different AML subtypes observed in patients with NUP98-NSD1 and NUP98-KDM5A fusions. We propose cbCD34+ HSPCs as a promising system for in vitro modeling of NUP98 fusions. Citation Format: Ryan Hiltenbrand, Jonathan Miller, Jeffery Klco. Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B34.

Abstract B16: MECOM dysregulation is associated with poor outcome in pediatric therapy-related myeloid neoplasms

Abstract Therapy-related myeloid neoplasms (tMN) occur in children as a consequence of cytotoxic therapies used to treat childhood malignancies, are typically resistant to conventional chemotherapies, require hematopoietic cell transplantation as the only curative option, and typically have a dismal prognosis. While the genomic alterations that drive tMN in children have yet to be comprehensively described, alterations involving the MECOM locus have been described in some myeloid neoplasms like tMN. Overexpression of MECOM is associated with poor prognosis in myeloid malignancies, is present in ~10% of adult cases, and occurs at an increased frequency in pediatric AML with KMT2A rearrangements (KMT2Ar). RNA sequencing from a cohort of 56 pediatric tMN cases collected at St. Jude shows that 24 (43%) cases within our cohort express high levels of MECOM (FPKM value &amp;gt;5). In these 24 cases, we identified one with a canonical fusion (RUNX1-MECOM), one with a NUP98 fusion (NUP98-HHEX), and 18 with KMT2Ar. Whole-genome sequencing demonstrated that two of the remaining MECOMHigh cases have a t(2;3)(p21;q26.2) involving MECOM on chromosome 3 and noncoding regions of chromosome 2 adjacent to ZFP36L2, a gene highly expressed in hematopoietic cells. Further, ENCODE data support that this region of the genome is an active enhancer, suggesting a proximity effect in which this enhancer has been hijacked to drive high levels of MECOM expression. The two remaining cases with aberrant and unexplained MECOM expression are currently under evaluation. In our cohort, MECOM expression levels are predictive of a worse outcome (overall survival at 2 years: MECOMHigh=12.5% vs. MECOMLow=40.6%; log rank p&amp;lt;0.01). Although KMT2Ar was frequently present in our cohort 28/56 (50%) and enriched in the MECOMHigh group (MECOMHigh=75% (18/24) vs. MECOMLow=31% (10/32; p&amp;lt;0.01), high MECOM expression did not confer a significant survival difference within the KMT2Ar group (overall survival at 2 years: MECOMHigh=16.7% vs. MECOMLow=40%; log rank p=0.33). In conclusion, we report a large cohort of pediatric tMN and show that high levels of MECOM expression predicts a worse outcome. Additionally, we identify enhancer hijacking as the mechanism through which at least a portion of high MECOM expression may be explained. Citation Format: Tamara Lamprecht, Jason R. Schwartz, Jing Ma, Michael P. Walsh, Jeffery M. Klco. MECOM dysregulation is associated with poor outcome in pediatric therapy-related myeloid neoplasms [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B16.