Abstract A19: Human models of multilineage leukemia induced by NUP98-KDM5A chimeric fusion oncogene
Authors: Cardin Sophie , Bilodeau Melanie , Laramee Louise , Jouan Loubna , Roussy Mathieu , Humphries R. Keith , Hebert Josee , Wilhelm Brian T. , Cellot Sonia 
Published: February 2019

Abstract
Acute megakaryoblastic (CD41+CD61+) leukemia (AMKL) is a subset (10%) of pediatric acute myeloid leukemia (AML) that typically affects young children (&lt;3 yrs) and carries dismal cure rates (&lt;40%), due to primary chemoresistance or early relapse. It was recently recognized that mutually exclusive chimeric fusion oncogenes are found in 60-70% of AMKL, including the NUP98 (nucleoporin 98)-KDM5A (histone lysine demethylase 5A) fusion, in ~10% of cases. AML subtype-specific human models are needed to identify subtype-specific biomarkers and drug targets, and to overcome the paucity of primary human samples, the genetic heterogeneity of the disease, and the clonal evolution of commercially available cell lines.
We engineered human models of AMKL through overexpression of NUP98-KDM5A (N5A) in cord blood (CB)-HSPC using lentiviral transduction (vector carrying GFP reporter gene). We generated multiple de novo N5A models, from distinct genetic backgrounds (independent CB units), to identify fusion-specific features. Adoptive transfer of the fusion oncogene-carrying cells (or N5A-cells) in immunodeficient (NSG) recipient mice led to AMKL development with a latency of 32-46 weeks (λ of 1/6 to 2/3, n=3 independent experiments). Mice presented clinically overt signs of disease with leukemic cell infiltration of multiple organs (bone marrow, liver, and spleen, as well as lung and kidney) and myelofibrosis. Leukemic blasts consisted of CD45loCD34-CD41+CD61+ cells, faithfully phenocopying human AMKL. In addition, N5A overexpression led to the development of acute erythroid leukemia (AEL) (CD34-CD117+CD36+) and B-ALL (CD34+CD38+CD19+CD10+), reflecting the clinical heterogeneity of leukemia associated with NUP98 fusions and suggesting distinct cells of origin for each entity. While N5A harboring B-ALL have not yet been reported, the fusion is now emerging as a recurrent genetic aberration in AEL. Transcriptomic studies of flow-sorted GFP+ xenograft cells closely matched that of N5A pediatric samples, and showed upregulation of HOXA/B cluster genes, as compared to control HSPC. A hematopoietic stem cell signature was detected by gene set enrichment analysis (GSEA) for N5A AMKL, as well as novel cell surface markers, including p-selectin (SELP) and CD96. Thus far, cooperating mutations were not identified, suggesting that the N5A fusion is a potent oncogene.
In addition, using optimized culture conditions to promote HSPC expansion, leukemic cell lines were generated. N5A-cells displayed maturation block and increased cell proliferation compared to controls, as assessed by cytology, serial tracking of increasing CD34+GFP+ cell fractions, and lack of a c-KIThi differentiated mast cell population. After 3 weeks in vitro, when control-transduced cells normally begin to die off, an immature cell population emerged from the N5A-cells and this population was sustained for &gt;140 days. This was observed by day 25 of culture in 66% (2/3) to 92% (11/12) of the wells (n=3 expts). Progenitor frequency was markedly increased in N5A-cells (on average 1:3 vs 1:200 in CTL, at day 88 of culture; n=3 expts). These N5A engineered cell lines can be efficiently transduced with shRNA carrying lentiviral vectors and thus provide suitable substrates for chemical and genetic screening approaches.
We have optimized a strategy to engineer human cell lines and xenograft models of high-fatality NUP98-KDM5A driven leukemia, identifying specific leukemia-activated genes and novel cell surface biomarkers for AMKL. These studies unraveled the unappreciated multilineage leukemic potential of N5A, indicating that engineered leukemia can be exploited to unravel novel disease subtypes, which is especially relevant for cryptic mutations such as N5A fusions. The generated cell lines pave the way to genetic- or chemical-based functional screens to identify promising drug targets.
Citation Format: Sophie Cardin, Melanie Bilodeau, Louise Laramee, Loubna Jouan, Mathieu Roussy, R. Keith Humphries, Josee Hebert, Brian T. Wilhelm, Sonia Cellot. Human models of multilineage leukemia induced by NUP98-KDM5A chimeric fusion oncogene [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A19.