Fig 2: FTY720 inhibited ANCA-induced neutrophil activation while upregulating FAO. (A) Identification of increased neutrophil marker CD11b of differentiated HL-60 cells. (B) Inhibitory effect of FTY720 on respiratory burst of differentiated HL-60 cells stimulated with serum from AAV patients. (C) Inhibitory effect of FTY720 on respiratory burst of isolated human neutrophils activated by MPO-ANCA positive IgG. (D) Gene expression of the top six upregulated genes in FAO in RNA sequencing was measured in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. (E) mRNA level of PPARα and protein expression of CPT1a and PPARα in differentiated HL-60 cells treated with the 10% serum from AAV patients and FTY720 or vehicle. Data are shown as means (s.d.). *P < 0.05, **P < 0.01, ***P < 0.001; ns: not significant. AAV: ANCA-associated vasculitis; ABCD3: ATP binding cassette subfamily D member 3; CPT1a: carnitine palmitoyltransferase 1A; ECHDC2: enoyl-CoA hydratase domain containing 2; FAO: fatty acid oxidation; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HSD17b4: 17β-hydroxysteroid dehydrogenase 4; MFI: mean fluorescence intensity; PPARα: peroxisome proliferator activated receptor α; ROS: reactive oxygen species; SESN2: sestrin 2; SLC27A2: solute carrier family 27 member 2

Fig 3: Phenotypic analysis reveals distinct characteristics of PUUV LDGs and PMNs. (A) UMAP visualization of single live CD56-/CD3-/CD19-/CD14-/CD15+/CD66b+ cells, displaying LDGs (in blue) and PMNs (in red) in a concatenated format, enabling an in-depth examination of their phenotypic differences. (B) Multigraph color mapping depicting each fluorochrome’s Mean Fluorescence Intensity (MFI) for selected markers, including CD66b, CD11b, LOX-1, CD16, CD15, HLA-DR, and CD33. (C) Bar graph illustrating the proportion of PMNs and LDGs expressing CD16.

Fig 4: Cord blood CD34 models recapitulate phenotypes of NUP98r leukemiaA. Experimental schema using cord blood CD34+ cell models (cbCD34). B. Colony-forming unit assays of cbCD34 models with empty control vectors or NUP98::NSD1, NUP98::KDM5A, or NUP98::HOXA9- expressing vectors. C. Cell growth assays of cbCD34 models in liquid culture. D. Flow cytometric analysis of cbCD34 models in liquid culture (top: CD34+, mid: CD11b+, bottom: CD41a+ population ratio:% in mCherry+ live cells). E. Principal component analysis (PCA) of RNAseq data from liquid culture. Colors indicate NUP98 fusions, and shapes indicate days after transduction. F. Heatmap showing expression of representative genes related to stemness or differentiation of hematopoietic cells. Colors of cells indicate expression levels normalized among samples, and genes are annotated on the left. G. Comparison of differentially expressed genes (DEGs) in each cbCD34 model compared with empty vector controls at day 42. Venn diagram showing overlaps of highly expressed genes in each model (mid) and GO term analyses of shared or specific DEGs are shown (left, right). Data was obtained from three biological replicates (different lots of cord blood). In B-D, statistical tests were performed by generalized linear mixed effect model with Poisson (B) and Gaussian (C-D) distributions followed by comparison with empty vector control and the Benjamini-Hochberg adjustment, asterisks indicating adjusted P-values *<0.05. Error bars indicate mean ± s.e.m. Abbreviations. FO: fusion oncoprotein, PDX: patient-derived xenograft, FDR: false-discovery rate.

Fig 5: Cooperating alterations and differentiation status affect sensitivity to menin inhibitionA. Experimental schema showing revumenib treatment. B. Relative cell growth of cbCD34 NUP98::KDM5A models with gRNA treated with revumenib (0.1–1μM) compared with DMSO controls. C. Flow gating (top), CD34+CD41a+ population (left), CD34−CD41a+ population (mid), and CD34−CD11B+ populations (right) among mCherry+ GFP+ mAmetrine+ live population at day 15. D. GSEA analyses between DMSO and revumenib-treated conditions, colors showing NES (top-left), comparison of expression changes between AAVS and RB1-gRNA1 conditions (right), and GO term analysis of changes enriched (difference of fold changes >1) in AAVS conditions (bottom-left). E. Relative cell growth of unedited cdCD34 NUP98::KDM5A (control, black), CHRF-288–11 (red) and ST1653 PDX (blue) treated with revumenib (0.1–1μM) compared with DMSO controls. F. Schematics illustrating cellular hierarchy of NUP98::KDM5A models. Data was obtained from three technical replicates using gRNA-transduced cells in Fig.6. One data point at day 15 was excluded for technical errors. Statistical tests were performed by generalized linear mixed effect model with Gaussian distribution followed by the Benjamini-Hochberg adjustment (B, E) or Student’s t-test by comparing gRNA conditions with AAVS controls (C). Asterisks indicating P-values or adjusted P-values *<0.05. Error bars indicate mean ± s.e.m.