CUX1 regulates genes associated with MDS and JMML gene signatures and PI3K/AKT signaling. Primary human CD34+ HSPC were transduced with shCUX1-A, shCUX1-B, or nonspecific control shRNA. After 7 days, GFP+ CD34+ cells were sorted and RNA collected for RNA-sequencing analysis, with 2 biological replicates. (A) CUX1 transcript levels after CUX1 knockdown as determined by RNA-sequencing. (B) The volcano plot shows gene expression changes after CUX1 knockdown. Red indicates differentially expressed genes with a 5% false discovery rate (FDR). The horizontal dashed line represents a nominal P value = .05. Vertical dashed lines indicate ± one-fold change. (C) Heat map of the 339 differentially expressed genes identified after CUX1 knockdown, with a 5% FDR. (D) Gene set enrichment analysis of RNA-sequencing data. Gene sets used were: “Quiescent” and “Proliferative”43 ; GO gene set from the MSig Database,42 −7/del(7q) MDS vs normal karyotype MDS,33 and JMML.34 Genes listed are those that had nominal P values of <.05 (CUX1 knockdown vs control) and were within the GSEA “Core enrichment” results. (E) Whole BM was collected from Cux1mid, Cux1low, or Ren control mice after 5 days of dox, cultured in serum-free media for 2 hours, and stimulated with serum for 20 minutes. Whole cell lysates were probed for phospho-AKT by western blot. One representative blot of 3 biological replicates is shown. (F) qRT-PCR results from LSK and myeloid progenitors sorted from the indicated mice. One of 3 biological replicates is shown.
