(E) Manifestation of Mac-1, as determined by circulation cytometry, in the BM-derived tumor cells (M/E p53WT and p53null, MFI,
(E) Manifestation of Mac-1, as determined by circulation cytometry, in the BM-derived tumor cells (M/E p53WT and p53null, MFI, .0001; n = 5). suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Collectively, these findings demonstrate the restorative utility of this new class of inhibitors to treat highly aggressive AML by Closantel targeting LICs. Introduction Acute myeloid leukemia (AML) is usually a clinically heterogeneous disease characterized by a multitude of gene mutations and chromosomal abnormalities, resulting in marked differences in responses and survival following chemotherapy. In particular, AML driven by translocations involving the mixed-lineage leukemia (MLL) gene represent an aggressive subtype associated with early relapse following chemotherapy.1 MLL translocations occur in >70% of pediatric and >10% of adult AML, which are associated with an intermediate to unfavorable prognosis depending on the translocation partner and the presence of additional cytogenetic aberrations.2 New approaches targeting epigenetic regulators associated with the MLL-fusion protein complex, eg, bromodomain and extraterminal proteins and DOT1L histone methyltransferase, are currently being investigated in phase 1 clinical trials.3-5 However, it was recently reported that bromodomain and extraterminal protein inhibitors failed to target the leukemia-initiating cell (LIC) population, and thus drug resistance emerged.6 Consequently, there is still an urgent need for new therapies to treat these and other aggressive AML subtypes. Here, we have tested the therapeutic efficacy of a novel inhibitor of RNA polymerase I (Pol I) transcription, CX-5461,7 in genetically altered mouse models of AML driven by MLL or AML1/ETO fusion proteins, and main patient-derived xenograft (PDX) models. In both murine and human AML, CX-5461 exhibited a remarkable single-agent efficacy. Unexpectedly, in Closantel addition to the previously characterized mechanism of action of CX-5461 including activation of p53,8 we observed a p53-impartial response including phosphorylation of checkpoint kinase 1/2 (CHK 1/2) associated with a G2/M cell-cycle defect and induction of myeloid differentiation in leukemic blasts. Analysis of the hematopoietic compartment discloses that CX-5461 reduces the LIC Closantel populace in p53 wild-type (WT) and null AML, thus decreasing the disease-initiating potential in vivo and their clonogenic capacity. Together, these studies suggest that Pol I transcription inhibition may represent a encouraging new approach to treat human AML by targeting the LIC impartial of functional p53. Experimental procedures Animal work was approved by the Animal Ethics Committees at the Peter MacCallum Malignancy Centre (E462), Australian National University or college (E2015/12), SA Pathology/Central Adelaide Local Health Network Animal Ethics Committee (#52/15), and Alfred Medical Research and Education Precinct (E/1563/2015/M). C57Bl/6 mice were purchased (Walter and Eliza Hall Institute or Australian Phenomics Facility) and NOD.Cg-Web U2AF1 site). Propidium iodide (PI) or 4,6-diamidino-2-phenylindole (DAPI) was added as cell viability staining. Cell death assays were performed in 96-well plates with 1 g/mL PI incubated for 15 minutes at room heat, and analyzed using the BD FACSVerse cytometer. Cell-cycle distribution was analyzed via 5-bromo-2-deoxyuridine (BrdU) incorporation. Apoptotic cell death was analyzed by Annexin V/PI staining as explained.8 Clonogenic assays in methylcellulose Colony formation of primary patient AML or green fluorescent protein-positive (GFP+)-murine tumor cells was analyzed in methylcellulose (human M4435 and mouse M3434; Stemcell Technologies) as explained.6 Histology, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and May-Grnwald Giemsa staining Tissues were fixed in 10% neutral buffered formalin, femurs decalcified, and paraffin wax embedded and cut (4 m sections). Sections were stained with hematoxylin and eosin and TUNEL performed. GFP+-sorted cells were cytospun (2 moments, 800 rpm), air-dried, and stained with May-Grnwald Giemsa (Grale Scientific). Slides were analyzed using an Olympus BX-61 and images were captured using SPOT Advanced software. Immunoblotting Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to immobilon-P (Millipore), blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 at room temperature, and incubated with primary (supplemental Table 3) then secondary antibodies before detection by enhanced chemiluminescence (GE Healthcare). Quantitative real-time polymerase chain reaction RNA was isolated per manufacturers instructions (Bioline or Qiagen). Complementary DNA was synthesized using Superscript III (Invitrogen) and random hexamer primers, and quantitative real-time polymerase chain reaction was performed using Fast SYBR Green Grasp Mix (Applied Biosystems), 100 mol/L primer on a StepOnePlus Real-time PCR System (Applied Biosystems). Computed tomography values were decided, normalized to housekeeping genes, and fold changes in expression calculated using the 2C computed tomography method. Primer sequences are outlined in supplemental Table 4. RNA sequencing Sequencing libraries were prepared using.