The human Smoothened inhibitor PF-04449913 induces exit from quiescence and loss of multipotent Drosophila hematopoietic progenitor cells

The efficient treatment of hematological malignancies as Acute Myeloid Leukemia, myelofibrosis and Chronic Myeloid Leukemia, requires the elimination of cancer-initiating cells and the prevention of disease relapse through targeting pathways that stimulate generation and maintenance of these cells. In mammals, inhibition of Smoothened, the key mediator of the Hedgehog signaling pathway, reduces Chronic Myeloid Leukemia progression and propagation. These findings make Smo a candidate target to inhibit maintenance of leukemia-initiating cells. In Drosophila melanogaster the same pathway maintains the hematopoietic precursor cells of the lymph gland, the hematopoietic organ that develops in the larva. Using Drosophila as an in vivo model, we investigated the mode of action of PF-04449913, a small-molecule inhibitor of the human Smo protein. Drosophila larvae fed with PF-04449913 showed traits of altered hematopoietic homeostasis. These include the development of melanotic nodules, increase of circulating hemocytes, the size increase of the lymph gland and accelerated differentiation of blood cells likely due to the exit of multi-potent precursors from quiescence. Importantly, the Smo inhibition can lead to the complete loss of hematopoietic precursors. We conclude that PF-04449913 inhibits Drosophila Smo blocking the Hh signaling pathway and causing the loss of hematopoietic precursor cells. Interestingly, this is the effect expected in patients treated with PF-04449913: number decrease of cancer initiating cells in the bone marrow to reduce the risk of leukemia relapse. Altogether our results indicate that Drosophila comprises a model system for the in vivo study of molecules that target evolutionary conserved pathways implicated in human hematological malignancies.


Drug administration to Drosophila larvae and phenotypic assay
Fertilized females of the different crosses were synchronized and let lay eggs for one hour. Newly hatched L1 larvae were collected, washed with water, and then divided in batches of 30 animals. The amount of either DMSO-or PF-04449913-containing medium has been increased day by day according to the progression of the larval instars of development: L1 25 µl, L2 50 µl, e-L3 100 µl, m-L3 100 µl. Then, larvae were collected and analyzed for the melanotic nodule phenotype at l-L3 or they were dissected at m-L3 and processed for immunofluorescence. Control larvae were identically handled to the ones exposed to drug. Each experiment was repeated at least three times and the shown data represent the average. Larvae were observed under a stereomicroscope to assess the presence of melanotic nodules. Since melanotic nodules may arise as a consequence of immune response against pathogens, for each trial and tested compound concentration, a concurrent DMSO control has been performed. The average phenotypic penetrance of control larvae ranged from 6% to 10%.

Gal4/UAS mediated expression
We set up appropriated and synchronized crosses at 29°C to induce expression of specific UAS transgenes under the control of the indicated Gal4 drivers. Progeny was allowed to grow until l-L3 (72 h AEH). Staged larvae were genotyped and selected according to the GFP fluorescence pattern of the constructs of interest and/or of the used GFP-balancer chromosomes. Larvae carrying the genotype of interest were observed under the stereomicroscope to assess the presence of melanotic nodules or were dissected to isolate the lymph gland then processed following the fixation/immunolabeling protocols

Lymph gland dissection and fluorescent immunolabeling
Lymph glands were dissected in PBS, kept on ice and then fixed for 30 minutes in 4% paraformaldehyde in PBS at room temperature. After 3 washes in PBS 1X pH 7.5, 0.3 % Triton (PBT 0.3%), lymph glands were rinsed for 30 minutes in PBT 0.3% and NGS 10% (Normal Goat Serum-Jackson Immunoresearch). The samples were incubated overnight at 4°C with the indicated primary antibodies in PBT 0.3% and NGS 10%. The day after the samples were washed three times in PBT 0.3% then incubated for 3 hours at room temperature with the secondary antibodies in PBT 0.3% -NGS 10% and the nuclei were labeled with Hoechst 33258 (Sigma) 2 µg/ml in PBS 1X. Tissues were mounted on slides under a coverslip using Fluormount. The anti-Smo mouse monoclonal antibody (supernatant diluted 1:300) developed by Beachy P.A [10,11], the anti-Antp mouse monoclonal 4C3 (supernatant diluted 1:400; deposited to the DSHB by Brower D.), the anti-Ptc (Apa 1) mouse monoclonal antibody (concentrated Mab diluted 1:1000, deposited to the DSHB by Guerrero, I. (DSHB Hybridoma Product Drosophila Ptc (Apa 1)) and the anti-Notch mouse monoclonal antibody raised against the Notch intracellular domain (N ICD , diluted 1:1000, deposited to the DSHB by Artavanis-Tsakonas, S., DSHB Hybridoma Product C17.9C6) were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. The secondary antibody Goat-Cy3-anti-mouse (Jackson Immunoresearch) was diluted 1:400. The Rabbit anti-Phospho-histone H3 (PH3-Upstate Biotechnology, was diluted 1:100.

Imaging, quantification of the lymph gland phenotype
For each experimental or control group of animals, 11-21 lymph gland primary lobes have been scanned using a Nikon A1R confocal laser-scanning microscope, equipped with a Nikon PlanApo 40× lens and captured using NIS Elements AR 3.10 software (Nikon). For each lobe either GFP (corresponding to lymph gland MZ in domeless-Gal4,UAS-GFP larvae, or to CZ in hemolectin-Gal4,UAS-GFP larvae) or Hoechst (corresponding to the whole primary lobe) signals have been acquired along the Z-axis. The whole Hoechst signal was acquired along the Z-axis corresponding on average to 15.01 µm in control larvae and to 17.995 µm in larvae fed with the compound. The step among images was between 0.90 and 1.10 µm. Each Z stack has been projected on a single plane and the total lobe area (Hoechst) or the area occupied by GFP+ cells has been calculated using the NIS Element software.

Quantification of the proliferating cells
Lymph glands from m-L3 larvae fed either with PF-04449913-or DMSO-medium were dissected and immunolabeled with the anti-PH3 antibody and counterstained with the nuclear dye Hoechst. The whole thickness of 15 primary lobes from each group of larvae has been scanned using a Nikon A1R confocal laserscanning microscope. Each Z stack has been projected on a single plane and the cells labeled bye anti-PH3 antibody has been counted.