c-MYB and PU.1 mutated in a high-risk AML patient and reversed expressed in AML patients. (A) Blast cells in PB and bone marrow of AML patients(400×, red arrows indicate myeloid blasts). (B) Proportion of PHA cells in the bone marrow of AML patients with PU.1G15R or PU.1G157R; c-MYBNRD-ins variants after treatment (400×, blue arrows indicate PHA cells, mean ± SE). (C) Schematic of a normal trilobed neutrophil, bilobed PHA cell, and nonlobed PHA cell (graphical elements sourced from https://www.biorender.com/). (D and E) Correlation analysis of c-MYB and PU.1 expression in acute myeloid leukemia (AML) patients. The Spearman?s and Pearson?s correlation coefficients of c-MYB and PU.1 expression in 671 AML (Spearman?s: P = 6.41e-33, R = ?0.44; Pearson?s: P = 3.16e-25, R = ?0.39) (D) and 162 AML (Spearman?s: P = 3.70e-14, R = ?0.55; Pearson?s: P = 5.45e-14, R = ?0.55) (E) patients were measured.

The number of immature neutrophils was significantly increased in c-mybhyper;pu.1G242D/G242D zebrafish embryos. (A and B) Accumulation of neutrophils in c-mybhyper;pu.1G242D/G242D embryos. WISH of cebpa, cebp1, and mpx expression at 5 dpf in wildtype, c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D zebrafish. The caudal hematopoietic tissue (CHT) is enlarged in the red box (200×), n ? 6. (C) qPCR results of 5 dpf embryos from wildtype, c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D (n >20, three biological replicate). (D?I) The neutrophil maturity and enzyme activity were decreased significantly in the c-mybhyper;pu.1G242D/G242D zebrafish. Granulocyte maturity was detected by SB staining (D), SB+ cell counts (F) and mean optical density (H) were calculated and compared at 5 dpf. The CHT is enlarged in the red box (200×). Green boxes show further enlarged regions focusing on a single cell (400×), n ? 13. Peroxidase activity was detected by DAB staining (E), DAB+ cell counts (G) and mean optical density (I) were calculated and compared at 5 dpf. Red boxes show a further enlarged region (400×), n ? 10.

The myeloid cell count increased in 3-mo-old c-mybhyper;pu.1G242D/G242D zebrafishKM with neutrophils showing Pelger?Huët anomaly?like changes. (A?C) PB cells and KM blood cells (A) in 3-mo-old adult fish stained with May-Grunwald/Giemsa [400×, (black triangles indicate blasts, green triangles precursors, blue arrows indicate normal band/kidney-shaped neutrophils, and red arrows indicate abnormally rounded neutrophils)]. Blood cell counts of PB (B) and KM (C) were calculated manually based on their morphology. The black asterisks indicate statistical difference (n = 12). (D) Morphology and counting ratio of neutrophils with different nuclear morphologies (400×, the red box shows abnormal round nuclear cells in c-mybhyper;pu.1G242D/G242D zebrafish, the blue box shows normal immature round nuclear cells from the other three genotypes n = 6). (E) High-resolution imaging (upper, 600×) and TEM morphology (Lower) observation showed PHA-like cells in c-mybhyper;pu.1G242D/G242D zebrafish. Blue, yellow, red, and green arrows indicate crystal granules, vacuolar granules, chromatin clumping, and nuclear membranes, respectively. All TEM images are magnification ×12,000 and 80 kV.

Pelger?Huët-like cells are a group of pathological neutrophils with poor differentiation. (A) Five subpopulations of neutrophils (GMP-Gs, GPs, Neutrophils-1, Neutrophils-2, and Neutrophils-3). (B) Temporal analysis of neutrophil in different subtypes (curve arrow indicating differentiation direction). (C) Detailed population diagram of neutrophils composition of wildtype, c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D. Cells are colored by their cell-type annotation and numbered according to the legend beside. (The blue line indicated Neutrophils-2 population, the yellow line indicated Neutrophils-1 population, and the purple line indicated Neutrophils-3 population.) (D) Enriched GO pathways found in the analysis of DEGs in Neutrophils-3 vs. other neutrophil groups. Gene Ratio means Number of Differentially Expressed Genes/Total Number of Genes in the Pathway. Gene Number means the absolute number of target genes annotated to each pathway. (E) The expression of genes involved in differentiation.

Increased mortality and sAML transformation in c-mybhyper;pu.1G242D/G242D zebrafish. (A) Survival curves of wildtype, c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D zebrafish (n ? 21). (B?D) PB cells and KM blood cells (B) in 1-y-old adult fish stained with May-Grunwald/Giemsa (400×, (black triangles indicate blasts, green triangles precursor, blue arrow normal band/kidney-shaped neutrophils, red arrows PHA-like neutrophils). PB (C) and KM (D) cell counts were manually calculated (n ? 20). (E) Proportion of neutrophils with different nuclear morphologies in the four genotypes (n ? 10). (F) c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D zebrafish progressed to AML in adulthood (400×, black cones indicate early myeloid progenitors, blue arrows indicate myeloid cells in PB). (G and H) Bar plot of blood cell classification counts in the PB and KM of 7- to 15-mo-old zebrafish, AML-like sample (blast cells > 20%) are numbered as 1# to 8#. Red boxes indicate c-mybhyper;pu.1G242D/G242D blasts that exceed 20% in either PB or KM. (I) Lcp immunofluorescence confirmed myeloid cell infiltration into muscle tissue (white arrows). (J) Transplantation of c-mybhyper;pu.1G242D/G242D KM blood cells into foxn1?/? recipients recapitulate AML and PHA-like cell phenotypes. (black triangles indicate blasts, green triangles precursor, red arrows PHA-like cell). (K) KM blood cell counts in recipients of wildtype or c-mybhyper;pu.1G242D/G242D KM (n ? 20). Numbers in (B, F, I, and J) show zebrafish with representative phenotypes out of the total examined.

Impact oflmnb1 expression on PHA-like Cells and AML development. (A) CRISPR/Cas9 targeted exon 7 of zebrafish lmnb1. The target sequence is in red, and the PAM sequence in green. The lmnb1 (?4/?4) line has deleted nucleotides, leading to a truncated protein missing the lamin tail domain. (B) lmnb1 mRNA expression in 3 dpf lmnb1 mutants and siblings, detected by qPCR. (C) PHA-like cells in 3-mo-old lmnb1 (?4/?4) zebrafish (blue arrows, green arrows, and red arrows indicate segmented, band/kidney-shaped and round nucleus neutrophil, respectively). (D) Comparation of neutrophils with different shape of nucleus in wt and lmnb1 (?4/?4). The black asterisks indicate statistical difference. (E) KM blood cell morphology in 3-mo-old c-mybhyper;pu.1G242D/G242D and c-mybhyper;pu.1G242D/G242D;lmnb1OE (blue arrows, green arrows, and red arrows indicate segmented, band/kidney-shaped and round nucleus neutrophil, respectively). (F) Comparation of neutrophils with different shape of nucleus in c-mybhyper;pu.1G242D/G242D and c-mybhyper;pu.1G242D/G242D;lmnb1OE. (G) Three 6-mo-old lmnb1KO F0 exhibited an AML-like phenotype in KM (black triangles indicate precursors, AML-like F0 zebrafish were numbered as #1, #2, and #3). (H) Calculation of different blood cell types in 6-mo-old wildtype and lmnb1KO F0 zebrafish. (I) The expression of lmnb1 in 3 AML-like lmnb1KO F0 KM blood cells was detected by qPCR. (J) Blasts increased in 3-mo-old lmnb1 (?4/?4) zebrafish (green arrows, red arrows, black triangles, and green triangles indicate lymphocytes, myelomonocytes, blasts, and precursors, respectively). (K) Calculation of different blood cell types in 3-mo-old wildtype and lmnb1 (?4/?4) zebrafish. (L) Calculation of different blood cell types in 3-mo-old c-mybhyper;pu.1G242D/G242D and c-mybhyper;pu.1G242D/G242D;lmnb1OE.

The c-mybhyper;pu.1G242D/G242D MDS/sAML model responds to the combination of all-trans retinoic acid and cytarabine treatment. (A) Flowchart for drug treatment in wt, c-mybhyper, pu.1G242D/G242D, and c-mybhyper;pu.1G242D/G242D embryos. (B) The combination treatment of Ara-C and ATRA decreased the number of neutrophils in the CHT region as determined by SB staining. (C) Average numbers of SB+ cells per embryo with drug treatment at 5 dpf (n ? 12).

Schematic of the synergistic effect of c-Myb hyperactivation and Pu.1 deficiency in promoting sAML transformation.