EVI-1 Mediates Apoptosis Resistance Via CD261 Induction and Enhances Leukemogenic Potential in Human Acute Lymphoblastic Leukemia

Abstract 1356

The transcriptional regulator ecotropic viral integration site-1 (EVI-1) has mainly been studied for its role in myeloid malignancies, in which high EVI-1 levels are associated with particularly aggressive disease. The role of EVI-1 in lymphoid cells, however, is largely unknown. Here, we show that EVI-1 is indeed expressed and functional in lymphoid malignancies. EVI-1 expression was analyzed by real-time PCR in 5 lymphoid cell lines and primary cells from patients with pediatric and adult acute lymphoblastic leukemia (ALL; n=32), chronic lymphocytic leukemia (CLL; n=19) and acute myeloid leukemia (AML; n=20) as well as healthy bone marrow (BM; n=3) as control. High EVI-1 expression was detected in 5/32 pediatric ALL, 2/3 adult ALL, 2/19 CLL and 2/20 studied AML samples, but in none of the healthy BM controls. Interestingly, high EVI-1 expression appeared associated with poor clinical prognosis (p=0.017) as previously reported in myeloid malignancies. Next, EVI-1 shRNA and non-coding shRNA lentiviruses were used to generate EVI-1 knockdown and control cells from NALM-16 and REH cells. Stably transduced knockdown EVI-1 and control lines were obtained and EVI-1 knockdown was confirmed on gene and protein level by real-time PCR and western blot as well as intracellular FACS analyses. Knock-down of EVI-1 expression suppressed cell growth and metabolic activity and lead to increased spontaneous apoptosis. Moreover, exposure to staurosporine, Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)- or Etoposide-induced apoptosis was significantly stronger in knockdown versus control cells, as shown by measurement of DNA-content, Annexin V and caspase-3/7 activity as well as western blot for activated caspase 3 and PARP. Molecularly, apoptotic response to TRAIL was mediated by induction of its receptor CD261 in EVI-1 knockdown cells, as shown by experiments performed with TRAIL and CD261 blocking antibodies. No significant proliferation differences were noted in cell cycle analyses and BrdU assays performed with EVI-1 knockdown and control treated NALM-16 and REH cells, which is in contrast to previous reports with mouse hematopoietic stem cells where EVI-1 regulates proliferation through Gata2.

Furthermore, in vivo leukemogenic capacity was assessed by transplantation of EVI-1 knockdown and control NALM-16 cells in immune-permissive NOD/SCID/IL2Rγ^null (NSG) mice. Inhibition of EVI-1 severely impaired leukemogenic properties of NALM-16 cells, resulting in suppression of leukemic engraftment rates and dramatic inhibition of organ infiltration by leukemic cells. Consistently, overall survival was prolonged in mice transplanted with EVI-1 knockdown versus control cells. Taken together, our data show that EVI-1 is not only expressed in myeloid but also lymphoid leukemias and might contribute to the leukemogenic potential and apoptosis resistance of ALL cells. Experiments underway in our laboratory are further investigating the molecular mechanisms by which EVI-1 regulates apoptosis sensitivity and putative differences between effects in cells of myeloid and lymphoid origin. Future studies in larger patient cohorts will be needed to explore the prognostic impact of EVI-1 expression in lymphoid neoplasia.