MDS1-EVI1 Complex (MECOM) Activation Promotes Leukemic Stem Cell Quiescence By Upregulating CDKN1C/P57Kip2

Background: Activation of MDS1-EVI1 Complex (MECOM) via proviral insertion at its gene locus (Morishita et al., 1988) or through chromosomal rearrangements at 3q26 (Suzukawa et al., 1994; Tang et al., 2019) results in marked overexpression of EVI1, and is predominantly associated with myeloid malignancies (Secker-Walker et al., 1995), especially high-risk acute myeloid leukemia (AML) (Barjesteh van Waalwijk van Doorn-Khosrovani et al., 2003). We previously showed that EVI1 overexpression skews hematopoiesis toward the myeloid lineage via the upregulation of a master regulator of myelopoiesis PU.1 encoded by SPI1 (SFPI1) (Ayoub et al., 2018), which explains the high cooccurrence of 3q abnormalities in myeloid malignancies. However, the role of EVI1 in reduced survival and high rates of relapse in AML patients is still unknown, and therapies specific for EVI1+ AML are absent due to the lack of EVI1 specific targets. Here we describe a novel mechanism of action of EVI1 in controlling leukemic stem cell quiescence by upregulating an essential member of the cyclin-dependent kinase inhibitor (cip/kip) gene family: CDKN1C/P57^Kip2.

Results: We first identified the upregulation of CDKN1C in the presence of EVI1 overexpression using RNA-seq on sorted hematopoietic stem and progenitor leukemia cells from our previously published EVI1+ leukemia mouse model (EVI1^TO) (Ayoub et al., 2018). Since CDKN1C/ P57 expression has been linked to high relapse rates in AML patients following chemotherapy (Radujkovic et al., 2016), we confirmed the upregulation of CDKN1C using RNA-seq on sorted populations (mononucleated cells (MNCs), leukemia stem cells (LSCs), and minimal residual disease (MRD)) from activated-MECOM AML patients in comparison to nonactivated-MECOM high risk AML patients (67 samples from 27 patients). Additionally, we found EVI1 overexpression produces a reversible block in differentiation and engraftment that can be rescued by ablating EVI1 overexpression or by additional mutations. In an effort to identify EVI1-induced transcriptional regulations for CDKN1C, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) and assayed for transposase accessible Chromatin (ATAC-seq) using our in vivo and in vitro EVI1 overexpression models. ChIP-seq for EVI1 showed a binding site for EVI1 located 300 kb from CDKN1C transcription start site (TSS), and ATAC-seq in EVI1 overexpressing AML showed an open chromatin pattern at the CDKN1C TSS only in the presence of EVI1 overexpression. We are presently investigating the molecular mechanism involved in the transcriptional regulation of CDKN1C in the presence of EVI1 overexpression.

Conclusions: Our data suggest a correlation between EVI1 and CDKN1C expression in high risk AML with 3q abnormalities and provide insights into a potential molecular mechanism for MECOM activation in controlling leukemic stem cell quiescence and resistance to chemotherapy by upregulating CDKN1C.