Human AML with PRDM16 rearrangments depends on SPI1 for leukemogenesis. (A) Flow cytometric analysis of 2 patient AML samples (patient 1, MDA14; patient 2, MDA16). (B) Quantification of PRDM16 expression by RNA-seq of primary human AML specimen with PRDM16 rearrangements (n = 4) compared with AML without PRDM16 rearrangement (n = 6). (C-H) The recipient mice transplanted with PRDM16-translocated human AML samples (MDA14 and 16, labeled as patient-derived xenograft [PDX]) had increased human CD45⁺ cells (C) and white blood cells (WBCs) (D) and development of AML (E) compared with mice transplanted with cord blood cells (CBs). The mice also exhibited increased human myeloid cells in the blood (F), splenomegaly (G), and immature blasts in the peripheral blood (PB; H). (I) PDX-derived AML cells were kept nonelectroporated (NC), electroporated with phosphate-buffered saline (PBS), electroporated with Cas9-RNP with a negative control (Ctrl) sgRNA (NCsg), or electroporated with 5 different sgRNAs against SPI1 (SPI1sg1-5). Immunoblotting and quantification show that deletion of SPI1 significantly reduced the protein expression of PU.1 as compared with Ctrl cells (NC, PBS, and NCsg). (J-K) SPI1 deletion by CRISPR/Cas9, as in panel I, attenuated the proliferation of human PRDM16-translocated AML cells compared with Ctrl cells (NC, PBS, and NCsg) (J) and increased the expression of FOG1 and GATA1 (n = 3) (K). Additional Ctrl includes AML cells edited for ENAM, a gene involved in amelogenesis. (L-M) Human AML cells were edited for ENAM or SPI1, transplanted, and analyzed 2 months later for the presence of human CD45⁺ cells in the PB (L) and bone marrow (BM; M). All data represent mean ± standard deviation. *P < .05, ***P < .001 by Student t test. B, B cell; M, myeloid cell; PLT, platelet; RBC, red blood cell; T, T cell.