Figure 4

Shh inhibition induces cycling of dormant leukemic progenitors. a. Heatmap from patient samples, unsupervised agglomerative hierarchical clustering of cell cycle pathway genes using RNA-Seq data from FACS-purified progenitors (CD34⁺CD38⁺lin⁻PI⁻) from 8 chronic phase (CP) and 9 blast crisis (BC) patient samples. Bold indicates prior treatment before sample collection (Additional file 1: Table S1). Red indicates over- and green, under-expression relative to the median RPKM (Log2 scale). Grey represents no expression. b. Network analysis of BC and CP progenitors revealed CDKN1A and CDKN2A as cell cycle hub. c. Representative FACS plots in bone marrow CD45⁺ cells after 14 days of vehicle or PF-04449913 treatment. d. Cell cycle analysis of bone marrow from BC CML engrafted mice after 14 days of vehicle (n = 4) or PF-04449913 (n = 4). Student’s t-test *p < 0.05 for both G_0` and G₁ population compared with vehicle treatment. e. GSEA analysis summary table obtained from RNA sequencing data comparing PF-04449913 treated engrafted mice (n = 4) to control (n = 4) (average 24.7-58.0 million mapped reads/sample). “Regulation of Cell Cycle” pathway was significantly decreased in PF-04449913 purified human progenitors derived from treated mice (family-wise p value =0.02). f. GSEA enrichment plot of “Regulation of Cell Cycle” pathway gene expression following in vivo LSC treatment with PF-04449913. The horizontal heatmap shows SAM score in descending order for all 13,850 genes (SHH pathway genes indicated as vertical black bars). g. Normalized gene expression values for the 18 genes in the core enrichment subset from the “Regulation of Cell Cycle” pathway. All the genes had a negative SAM score and are sorted in order of descending SAM score along the x-axis. This order agrees with the order in the GSEA enrichment plot, where expression levels for these genes are significantly reduced in the PF-04449913 treated mice.