ETV6 Contributes to Maintenance of Leukemia Stem Cells in Acute Myeloid Leukemia with High EVI1 Expression

Aberrant expression of Ecotropic viral integration site 1 (EVI1), seen in approximately 10% of acute myeloid leukemia (AML) patients, contributes to development of AML, and is associated with chemoresistance and poor clinical outcome. While it is known that AML cells have a differentiation hierarchy like that of normal hematopoietic cells, and that leukemia stem cells (LSCs) at the top of the hierarchy are considered resistant to chemotherapy. Therefore, eradication of LSCs is important to achieve a cure for AML. EVI1 is known to be associated with stem cell features in AML, however, the function of EVI1 in the maintenance of LSCs has yet to be elucidated fully. In this study, we tried to clarify the molecular mechanism of LSCs maintenance in AML with high EVI1 expression.

First, we investigated the profiles of LSCs characterized by EVI1. For this purpose, we used murine EVI1-AML model, where 5-fluorouracil treated bone marrow cells were retrovirally transduced with FLAG-tagged EVI1 followed by transplantation into sub-lethally irradiated mice. In EVI1-AML mice, c-kit positive leukemia cells have a high leukemia reconstitution capacity compared to c-kit negative leukemia cells, which indicated that LSCs were enriched in the c-kit positive fraction. To investigate the LSC specific features, we compared gene profile of c-kit positive and negative EVI1-AML cells by RNA-sequencing and found that 2955 genes are upregulated in the c-kit positive EVI1-AML cells. Moreover, to find the downstream target of EVI1, we also performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) of EVI1 by anti-FLAG tag antibody in the c-kit positive EVI1-AML cells and found that 6653 genes are annotated from the EVI1 binding regions. From these results, 803 overlapping genes were extracted as candidate genes involved in the maintenance of EVI1-AML.

Then, in order to identify the key mechanism of maintaining LSCs in EVI1-AML, we performed in vivo clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) knockout screening targeting these 803 genes. We employed murine Cas9 expressing EVI1-AML model, and Cas9-expressing c-kit positive EVI1-AML cells were lentivirally transduced with the library containing 4 single guide RNAs (sgRNAs) per gene plus 50 non-targeting sgRNAs and then transferred into sub-lethally irradiated wild-type mice. After AML development, DNA samples from the c-kit positive and negative subpopulations were deep-sequenced with sgRNA abundance, and genes counted more frequently in the c-kit negative fraction were listed as candidates for maintenance of EVI1-AML.

Among these candidate genes, deletion of ETS variant transcription factor 6 (Etv6) in the c-kit positive EVI1-AML cells reduced the proportion of c-kit positive cells and suppressed proliferation in vitro, so we focused on ETV6 as the candidate gene for maintenance of LSCs in EVI1-AML. We confirmed that knockdown of EVI1 by short hairpin RNA in c-kit positive EVI1-AML cells reduced the expression level of Etv6, suggesting that ETV6 is a downstream target of EVI1. Then, in order to assess the ETV6 functions in LSCs in vivo, we performed a competitive transplantation experiment and found that Etv6 deletion in c-kit positive EVI1-AML cells reduced the ability to reconstitute leukemia in sub-lethally irradiated mice. We also found that Etv6 deletion in c-kit positive EVI1-AML cells delayed AML development in sub-lethally irradiated mice. These results suggest that ETV6 is necessary to maintain leukemia initiating potential in EVI1-AML.

In conclusion, we identified that ETV6 would be a downstream target of EVI1 and contribute to maintenance of LSCs in AML with high EVI1 expression.