USP22 protects PU.1 from proteasomal degradation. (A) Analysis of USP22 and H2Bub protein expression by western blot in myeloid progenitors. p84 was used as a loading control. (B) PU.1 expression in KM and KMUKO CD11b⁺Gr1^low cells by flow cytometry. (C) USP22 and PU.1 interaction in HEK293 cells overexpressing myc-USP22 and Flag-PU.1. (D) USP22 and PU.1 endogenous interaction in cells isolated from KM mice. (E) PU.1 ubiquitylation in HEK293 cells expressing myc-USP22 WT or C185A mutant. (F) PU.1 protein stability in HEK293 cells overexpressing Flag-PU.1 and myc-USP22. (G) CD11b⁺Ly6G⁺ expression in cKit⁺ KMUKO cells transduced with murine stem cell virus retrovirus control or PU.1 and cultured in methylcellulose with GM-CSF (10 ng/mL) for 7 days. (H) GSEA showing that genes upregulated in KMUKO progenitors were highly correlated with genes associated with pediatric AML with poor prognosis. (I) Inverse correlation between genes downregulated in KMUKO progenitors and genes associated with pediatric AML with good prognosis. (J) Overview of USP22 function in Kras-induced MPNs: Kras activation leads to increased proliferation and differentiation of myeloid cells. Absence of USP22 blocks myeloid differentiation that results in the accumulation of GMPs and myeloblasts, leading to AML. At the molecular level, USP22 interacts and deubiquitylates PU.1, increasing its protein stability. This results in the expression of genes necessary for the differentiation of myeloid cells. In the absence of USP22, the levels of PU.1 protein and its target genes decrease, leading to a differentiation block and leukemic transformation.