DEK-NUP214 Acute Myeloid Leukemia Depends on CRM1 In Vivo

Introduction: Acute myeloid leukaemia (AML) with the translocation DEK-NUP214 (t(6;9)(p23;q34)) is a rare subtype present in 1-2% of AML patients. This translocation is associated with poor clinical outcome, resistance to chemotherapy and remains poorly characterized. Among all nucleoporins, NUP214 has the highest affinity for CRM1, a major exportin protein. We hypothesized that inhibition of CRM1 would be an effective treatment against the aberrant activity of DEK-NUP214. We assessed the leukemia cell line FKH-1, which has a DEK-NUP214 translocation as its sole mutation. In parallel, we established a patient-derived xenograft (PDX) model with the intent to further characterize the disease and assess the efficacy of CRM1 inhibitor KPT-8602/eltanexor.

Methods: Cell viability, induction of apoptosis, cell cycle and proliferation were measured at 24h, 72h and 96h for FKH-1 cells after single treatment with eltanexor and cytarabine. Changes in gene expression were assessed by real time PCR. Confocal immunofluorescence microscopy was used to study the localization of target proteins.

Leukemic blasts from five DEK-NUP214 positive patients were injected intravenously into irradiated NSGS mice. Engraftment of human cells in peripheral blood was monitored monthly by flow cytometry, and engrafting cells were serially transplanted. The DEK-NUP214 fusion transcript was confirmed by Sanger sequencing and molecular aberrations were evaluated with a custom myeloid sequencing panel.

Five weeks after transplantation, treatment with eltanexor or vehicle (CTL) was initiated in 5 mice per group at 12.5 mg/kg (for 12 weeks at 5 days/week, and 4 weeks at 4 days/week).

Results: Eltanexor treated FKH-1 cells showed an increase in apoptotic cells in a dose-dependent manner (42.4% at 100 nM vs 23.4% with CTL, p=0.0008), a decrease in cell proliferation, a reduction in overall cell viability, and downregulation of the fusion gene as well as EYA3, PRDM2 and SESN1, which are putative target genes of DEK-NUP214. Although cytarabine treatment induced apoptosis in FKH-1 cells, the downstream targets were not affected, suggesting an on-target effect of eltanexor. Eltanexor inhibited cell cycle (proportion of cells in S phase for eltanexor 1.02% at 400nM vs 7.51% for CTL, p=0.018) and disrupted the co-localization of CRM1 and DEK-NUP214 in the nucleus. In addition, eltanexor reduced the formation of DEK-NUP214 nuclear bodies and reduced protein expression of CRM1 in whole cell lysates.

In the first PDX model (PDX1), primary and secondary transplantations showed a mean of 12.5 and 43.9% of CD45+CD33+ cells in peripheral blood at 20-24 weeks. PDX1 mice had high white blood cell counts, low platelets and hemoglobin at time of sacrifice, as well as increased spleen weight and bone marrow blasts, which confirmed the development of AML. The engrafted cells showed a myeloid phenotype with expression of CD38, CD33 and CD14, with higher engraftment in bone marrow than spleen. Mutational analysis showed that the PDX samples retained the same mutations as observed in the patient sample (FLT3-ITD and WT1) besides the DEK-NUP214 fusion transcript.

The eltanexor or CTL in vivo treatment started 5 weeks after transplantation similar mean leukemic engraftment between treatment and control groups. After sixteen weeks of treatment and additional 5 weeks of observation, CTL treated mice developed leukemia with 19% engraftment of leukemic cells in peripheral blood, while eltanexor treated mice remained negative, p=0.003. This was accompanied by a decrease in WBC, hemoglobin, and platelet count in CTL treated mice, while the eltanexor treated group retained normal counts. Forty two weeks since transplantation, four vehicle mice have succumbed to disease while eltanexor treated mice remain leukemia free. Gene set enrichment analysis (reactomeDB) from RNAseq data of treated PDX1 cells showed upregulated neutrophil degranulation, signaling by interleukins, specifically IL-10, IL-4, and IL-13, as well as reduced activation of anterior HOX genes in eltanexor vs vehicle treated mice.

Conclusion: DEK-NUP214 positive FKH-1 cells are sensitive to CRM1 inhibition by eltanexor, which downregulates putative gene targets of DEK-NUP214 leukemia. We have successfully established a DEK-NUP214 PDX model and show exceptional sensitivity of these cells to eltanexor in vivo.

Landesman:hC Bioscience, Inc: Current Employment; Karyopharm: Ended employment in the past 24 months. Döhner:Agios: Research Funding; Astellas: Research Funding; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kronos: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Glycostem: Consultancy, Research Funding; Kura Oncology: Consultancy; Pfizer: Consultancy, Research Funding; PinotBio: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; BMS: Consultancy; Agios: Consultancy, Research Funding; Takeda: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Eurocept: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Tolremo: Consultancy; Astellas: Research Funding; Bayer Pharma AG: Research Funding; BergenBio: Research Funding; Loxo Oncology: Research Funding.