NUP98-KDM5A Fusion Induces Hematopoietic Cell Proliferation and Alters Myelo-Erythropoietic Differentiation
Authors: Miller Jonathan , Hiltenbrand Ryan , Lamprecht Tamara , Seth Aman , Abdelhamed Sherif , Iacobucci Ilaria , Mullighan Charles G. , Klco Jeffery M. 
Published: November 2019

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98 gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novo cases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5A is further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5A on the growth and differentiation of human hematopoietic cells has not been systemically studied.

In this study, we transduced cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) with NUP98-KDM5A or control lentiviral vectors and examined cell proliferation and differentiation. Exposure to cytokines (SCF, TPO, IL-6, FLT3L, SR-1) selected for CD34+ cell growth, and expression of NUP98-KDM5A increased proliferation rate in liquid culture by 19.4-fold (p<0.01). In addition to a growth advantage, expression of NUP98-KDM5A led to differences in differentiation. NUP98-KDM5A expressing cells showed a 4.9-fold decrease (p<0.05) in myeloid terminal differentiation (defined as the CD33+, CD11B+ population) and 2.8-fold increase (p<0.05) in early stem cell progenitors (defined as the CD117+ population). Interestingly, NUP98-KDM5A expressing cells showed a 3.3-fold increase (p<0.01) in the erythroid population (defined by the CD235a+, CD71+ population). The increase in erythroid differentiation was confirmed by RNA sequencing, which showed an enrichment in genes involved in heme metabolism (Hallmark Heme Metabolism, FDR q-value 7.33x10-8) after only 72 hours post-transduction. Furthermore, RNA sequencing demonstrated upregulation of HOXA genes, including HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10 and HOXA11 (p 3.36x10-2 - 1.16x10-7), which has been previously described in other high-risk leukemic fusions (including other NUP98 fusions) as a primary mechanism of stem-cell renewal.

To further elucidate NUP98-KDM5A effects on differentiation, cytokine supplements known to drive myeloid differentiation (SCF, TPO, G-CSF, and GM-CSF) were added to culture media, which demonstrated the same disruption in myelo-erythropoiesis based on a 4.0-fold decrease (p<0.001) in the terminal myeloid population, a 3.2-fold increase in CD117+ (p<0.001), and 2.6-fold increase in erythroid markers (p<0.001). Furthermore, NUP98-KDM5A recapitulated this phenotype in methylcellulose colony forming assays, with increased BFU-E and reduced CFU-GM colonies compared to control (p<0.01), despite no difference in overall colony number. Taken together, these in vitro studies demonstrate increased proliferation and altered differentiation in HSPCs by NUP98-KDM5A, with the pattern of differentiation by NUP98-KDM5A paralleling the M6 (erythroleukemia) AML subtype observed in patient samples harboring this fusion, supporting the clinical observation that NUP98-KDM5A is often a monogenic driver.

To evaluate the leukemic potential of NUP98-KDM5A, cells were injected into NRG-SGM3 mice with NUP98-KDM5A inducing at 12 weeks a rapidly lethal myeloid disease in vivo (97.8% human CD45+ cells vs. 2.3% in control, p<0.001). Furthermore, spleens of the NUP98-KDM5A mice were markedly enlarged, weighing on average 926.7g vs. 47.3g spleens seen in control, a 19.8-fold increase in weight (p<0.05).

In conclusion, the expression of NUP98-KDM5A in HSPCs drives proliferation and alters differentiation to maintain stem cell markers while driving an erythroid phenotype in vitro. Furthermore, this fusion is sufficient to engraft in the bone marrow and spleen of NRG-SGM3 mice in vivo and cause marked splenomegaly. Taken together, these data suggest this model properly recapitulates the human disease phenotype seen in patients expressing NUP98-KDM5A and future functional assays and drug screens may provide important insights into understanding the pathophysiology and pharmacologic vulnerabilities of this fusion class.

Disclosures

Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.