MLL-driven leukemias are sensitive to inhibition of hyperactivated Pol I transcription in vitro. (A) Pol I transcription is upregulated in malignant myeloid (M/E, GFP⁺-sorted) cells compared with normal myeloid (Mac-1⁺) cells from the bone marrow (BM) of C57Bl/6 mice. rRNA FISH with DAPI counterstain in malignant myeloid (GFP⁺) cells compared with normal myeloid (Mac-1⁺) cells. Images were taken with a ×63 objective. (B) Culture-adapted MLL-driven AML cells (M/E and M/A9) (M/E either p53WT or null) were treated with vehicle or CX-5461. The cells were treated with increasing concentration of CX-5461 for 1 hour, and 47S precursor rRNA synthesis was analyzed by ³²P-orthophosphate labeling (average IC₅₀ of 100 to 200 nM in p53WT and 400 to 500 nM in p53null AML). Graph represents mean ± SEM; n = 3. (C) Cell death was determined by PI incorporation after 24 hours of CX-5461 treatment (M/E IC₅₀ = 280 nM; M/A9 IC₅₀ = 37 nM; M/E p53null IC₅₀ = 5328 nM; and M/E p53null IC₅₀ = 3800 nM). Graph represents mean ± SEM; n = 3. (D) Apoptotic cell death was analyzed by Annexin V/PI staining using flow cytometry, after cells were treated with 100 nM and 1000 nM CX-5461 for 24 hours. Representative dot plots from n = 3. (E) Western blot analysis of total p53 and p21 protein after M/E and M/A9 p53WT cells were treated with 100 nM CX-5461 for the times indicated. Representative western blot from n = 3. a.u., arbitrary units; IC₅₀, 50% inhibitory concentration; SEM, standard error of the mean.