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Chromosomal translocations are often found in acute leukemia and frequently result in the generation of fusion proteins with oncogenic properties. We recently studied the immune evasion properties of PML-RARA and AML1-ETO, two common oncogenic fusion proteins in acute myeloid leukemia (AML). We found that both of these fusion proteins downregulate the expression of CD48, a ligand of the NK cell-activating receptor 2B4, thus leading to impaired NK cell cytotoxicity. However, it remained unclear whether other leukemic fusion proteins can manipulate NK cell ligands. To explore this issue, here we tested the effects of several leukemic fusion proteins on the expression of NK cell ligands. To examine whether NK cell ligands are downregulated by oncogenic fusion proteins other than PML-RARA and AML1-ETO, we cloned several oncogenic fusion proteins associated with acute leukemia into lentiviral vectors. The oncogenic fusion proteins we examined were MLL-AF4, NUP98-HOXA9, DEK-NUP214, and CBFBSMMHC. MLL-AF4 is associated with acute lymphoblastic lymphoma, whereas the other three fusion proteins are associated with AML. We expressed these fusion proteins in U937 cells (since this is the only cell line that could be transduced and this cell line is commonly used to express leukemic fusion proteins). We confirmed the expression of each of these fusion proteins in U937 cells by qPCR (data not shown). Expression of MLL-AF4, NUP98-HOXA9, and DEK-NUP214 did not affect the level of several NK cell ligands including the NKG2D ligands MICA, MICB, ULBP1, ULBP2, and ULBP3 (Supplementary Figure S1). The expression of B7-H6, MHC class I (Supplementary Figure S1), and CD48 (Fig. 1a) was also not affected. By contrast, the expression of the fusion protein CBFB-SMMHC led to a nearly complete abolishment of CD48 expression while not affecting the other NK cell ligands (Fig. 1b). Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4–CD48 interaction. We found that cells that express CBFB-SMMHC were killed significantly less than the control cells (Fig. 2a). To verify that the killing was indeed mediated by CD48, we blocked CD48 on the target cells and observed almost no killing of any of the targets (Fig. 2b). We also tested the killing of CBFB-SMMHC-expressing cells by IL-2-activated primary bulk NK cells and found that cells expressing the CBFB-SMMHC fusion protein were killed significantly less than cells that expressed an empty vector (Fig. 2c). The blocking of CD48 on the target cells significantly reduced the killing of all cells to a similar extent (Fig. 2d). Hence, the downregulation of CD48 by CBFB-SMMHC is functional and leads to reduced NK cell-mediated killing. To test the mechanism by which CBFB-SMMHC influences CD48 expression, we first tested the mRNA levels of CD48 by qPCR after overexpression of this fusion protein. We found that overexpression of CBFB-SMMHC reduced the mRNA levels of CD48 (Supplementary Figure S2). Next, we tested whether the effect of the CBFBSMMHC protein on the expression of CD48 depends on recruitment of histone deacetlyase (HDAC). We generated two deletion mutants of CBFB-SMMHC, which have

The oncogenic fusion protein CBFB-SMMHC downregulates CD48 to evade NK cell recognition