Aberrant RNA Splicing Contributes to the Pathogenesis of EVI-Rearranged Myeloid Leukemias
Authors: Inoue Daichi , Lee Stanley C , Yoshimi Akihide , Taylor Justin , Bitner Lillian E , Tanaka Atsushi , Hayashi Yasutaka , Cho Hana , Fukui Chie , Saganty Ruth , Penson Alexander V , Abdel-Wahab Omar 
Published: February 2020

Inversions between chromosome 3q21 and 3q26 ("inv(3)/t(3;3)") mark an aggressive, poor prognosis form of AML with limited treatment options and also occasionally occur in MDS and CML. Inv(3)/t(3;3) repositions a distal GATA2enhancer from 3q21 to the EVI1locus at 3q26 inducing ectopic EVI1 expression and reducing GATA2 expression. At the same time, additional genomic alterations exist in inv(3)/t(3;3) leukemias but the contributions of these events to inv(3)/t(3;3) leukemia are not well understood.

Here we evaluated genomic alterations in 63 patients with inv(3)/t(3;3) leukemia. Mutations in NRAS and the core RNA splicing factor SF3B1 were the most common individual alterations (each occurring in 27% of patients; Fig.A). We next quantified gene expression and splicing in AML samples with and without inv(3)/t(3;3) abnormalities and SF3B1 mutations. Amongst inv(3)/t(3;3) AML, the majority of gene expression changes were driven by the inv(3)/t(3;3) rearrangement while the majority of splicing changes were driven by mutant SF3B1. The most abundant category of splicing change in inv(3)/SF3B1 co-mutant cells was alteration in 3' splice site usage (Fig.B). Intriguingly, one of the most robust changes in splicing in inv3/SF3B1 co-mutant cells was aberrant splicing of EVI1itself such that SF3B1 mutant cells promoted expression of a novel isoform of EVI1using an intron proximal 3' splice site (Fig.C; the official gene name "MECOM" corresponds to the genes MDS1and EVI1at this locus).

While several mRNA isoforms of EVI1have been previously described, these all result in loss of EVI1 functional domains. In contrast, the unique EVI1 isoform in SF3B1 mutant/inv(3) AMLs contains in an in-frame insertion of six amino acids in the second zinc finger domain (ZF2) of EVI1 (a region of EVI1 known to be affected by germline mutations in leukemia predisposition syndromes). These data identify that nearly one-third of inv(3) AML patients express a heretofore undescribed isoform of EVI1. Of note, this unannotated EVI1isoform is also present in EVI1expressing/SF3B1K700Emutant leukemias lacking inv(3)).

The above findings highlight a novel model where inv(3)/t(3;3) AML is driven by ectopic expression of distinct oncogenic isoforms of EVI1. To test this model and understand the contribution of SF3B1K700Eto inv(3)/t(3;3) AML, we crossed transgenic mice bearing the entire human inv(3)(q21;q26) locus whereby the GATA2enhancer misdirects human EVI1expression ("inv3 mice"; Yamazaki et al. Cancer Cell2014) to Sf3b1K700Econditional knockin mice (Mx1-cre Sf3b1K700E/WT).Given that the human MECOMlocus (coding and noncoding regions) was recapitulated in this mouse model, the concordant novel EVI1isoform was expressed in inv3/Sf3b1K700Emice as in patients. While Mx1-cre Sf3b1K700Emice develop an MDS-like disorder, inv3 mice develop myeloid and lymphoid leukemias with lethality ~300 days after birth. However, expression of the Sf3b1K700E/WTmutation in inv3 hematopoietic cells resulted in a highly penetrant MDS, which transformed to a lethal AML by a median of 241 days (Fig.D;p=0.0021). In the first 6 months following transplant, Mx1-cre inv3 Sf3b1K700Emice had leukopenia, macrocytic anemia, and morphologic dysplasia (Fig.E-F) that eventually transformed to a disease with a high WBC count and large numbers of immature cells around time of death. In competitive reconstitution assays, Mx1-cre inv3 Sf3b1K700Ehematopoietic stem cells (HSCs) failed to differentiate into mature peripheral blood cells despite having a competitive advantage at the level of HSCs (Fig.G-H).

RNA-seq of hematopoietic precursors from the above models identified (i) a substantial change in splicing in inv3/Sf3b1K700Emutant leukemias versus those driven by inv3 alone, and (ii) alterations in a host of RNA binding proteins in inv3/Sf3b1K700Emutant leukemias (Fig.I).

These data highlight a high occurrence of SF3B1 mutations in inv(3)/t(3;3) leukemias, present a new genetically accurate model for inv(3) AML, and uncover a novel oncogenic isoform of EVI1 expressed in a large proportion of inv(3)/t(3;3) patients. Ongoing work focused on identifying the mechanistic effect of the SF3B1-mutant induced aberrant EVI1isoform may provide novel insight into the role of EVI1 in promoting leukemogenesis and engender development of therapeutic opportunities targeting EVI1splicing.

Figure

Disclosures

No relevant conflicts of interest to declare.