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. 2022 Feb 1;15(2):dmm048953.
doi: 10.1242/dmm.048953. Epub 2021 Nov 19.

Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes

Affiliations

Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes

Jun-Yi Zhu et al. Dis Model Mech. .

Abstract

Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. We have developed a Drosophila acute myeloid leukemia model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. We performed both genetic and drug screens using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed that echinomycin, an inhibitor of HIF1A, can effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, we showed that echinomycin treatment can effectively suppress oncogenic RAS-driven leukemia cell proliferation, using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes. This article has an associated First Person interview with the first author of the paper.

Keywords: Drosophila; Echinomycin; HIF1A; Hypoxia pathway; Leukemia; Mouse xenografts; Oncogenic RAS.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Human KRASG12V mutant transgene drives abnormal hemocyte proliferation in a Drosophila leukemia model. (A) Transgenic third-instar larvae carrying hemocyte-specific Hml-Gal4 driver directing expression of UAS-GFP (Control), and UAS-GFP plus UAS-HsKRASG12V (KRASG12V). Panels show stereo micrographs of larvae (left) and GFP fluorescence micrographs (right). Scale bar: 0.3 mm. (B) Hemocytes in hemolymph samples of equal volume extracted from control and KRASG12V third-instar larvae. Scale bar: 50 μm. (C) Quantification of total hemocytes per control and KRASG12V third-instar larvae (n=6; results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test). (D) Frequency plots comparing control and KRASG12V third-instar larval hemocyte cell size. Forward scatter (FSC) is a measurement of the amount of the laser beam that passes around the cell, which gives a relative size of a cell. (E) Frequency plots comparing control and KRASG12V third-instar larval hemocyte cell structure. Side scatter (SSC) is a measurement of the amount of the laser beam that bounces off particulates inside the cell, which is an indicator of granularity in a cell.
Fig. 2.
Fig. 2.
Human KRASG12V transgene expression alters the distribution of hemocyte subtypes and drives abnormal cell population expansion after transplantation. (A) Pro-hemocytes were selectively immunolabeled with anti-Wingless (Wg) antibody. The percentage of pro-hemocytes in total hemocyte is increased ∼1.5- to 2-fold in KRASG12V larvae (quantification shown below; n=6; results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test). Scale bar: 30 μm. (B) Crystal cells were selectively immunolabeled with anti-Lozenge (Lz) antibody. The percentage of crystal cells in total hemocytes is increased ∼2-fold in KRASG12V larvae (quantification shown below; n=6; results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test). Scale bar: 30 μm. (C) Plasmatocytes were selectively immunolabeled with anti-P1 antibody. The percentage of plasmatocytes in total hemocytes is reduced ∼10% in hemocytes KRASG12V larvae (quantification shown below; n=6; results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test). Scale bar: 30 μm. (D) Schematic showing larval hemocyte transplantation into adult flies. Hemocytes were collected from control (Hml-Gal4, UAS-GFP) or KRASG12V (Hml-Gal4, UAS-GFP; UAS-KRASG12V) third-instar larvae, then injected (500 hemocytes) into adult flies. Injected flies were maintained for 4 days, at the end of which time the hemolymph was collected and analyzed microscopically for hemocyte density. Lower panels show fluorescence micrographs of control compared to KRASG12V hemocytes (expressing GFP) 1 day after transplantation (day 1) and day 4 post-transplantation into adult flies. Scale bar: 50 μm. (E) Quantification of transplanted hemocyte numbers at day 1 and day 4 post-transplantation (results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test).
Fig. 3.
Fig. 3.
Human KRASG12V transgene expression is associated with Drosophila hemocyte immune function deficits. (A) Hemocytes (green) from control (Hml-Gal4, UAS-GFP) and KRASG12V (Hml-Gal4, UAS-GFP; UAS-KRASG12V) third-instar larvae co-incubated with fluorescent pHrodo Red-tagged S. aureus or E. coli (red). Scale bars: 10 μm. (B) Quantification of phagosome area in control and KRASG12V hemocytes co-incubated with fluorescent pHrodo Red-tagged S. aureus or E. coli (n=100; results presented as mean±s.d.; *P<0.05; unpaired Student's t-test). (C) Survival curves of control and KRASG12V adult flies infected with pathogenic P. luminescens bacteria or sterile PBS (vehicle) at 18°C. Forty flies were analyzed for each group. (D) Quantitative RT-PCR showing the relative expression level of genes involved in antibacterial defense in hemocytes from control compared to KRASG12V flies (n=3 replicates in each group; results presented as mean±s.d.; *P<0.05; unpaired Student's t-test). CecC, Cecropin C; modSP, modular serine protease; grass, Gram+-specific serine protease; LysX, Lysozyme X.
Fig. 4.
Fig. 4.
Drosophila KRASG12V genetic screen and the hits that rescue the hemocyte overproliferation and developmental lethality phenotypes. (A) Schematic illustration of the approach used for the genetic screen (see Materials and Methods section for a detailed description of rationale, strategy and procedure). (B) The 24 Drosophila genes that, when silenced in hemocytes, completely rescued KRASG12V-induced hemocyte overproliferation and developmental lethality phenotypes. Also listed are the human homologs of these genes, their DIOPT score (indicating the degree of conservedness) and the predicted function of the proteins encoded by these genes.
Fig. 5.
Fig. 5.
Silencing Drosophila HIF1 complex gene homologs sima and tgo rescues KRASG12V-induced hemocyte overproliferation, immune deficiency and pupal-stage developmental lethality. (A) Left upper and lower panels show control (Hml-Gal4, UAS-GFP) and KRASG12V (Hml-Gal4, UAS-GFP; UAS-KRASG12V) third-instar larvae, respectively, in which all hemocytes express GFP (green fluorescence). Middle and right panels illustrate the effects of silencing HIF1A homolog sima (sima-IR) and ARNT homolog tgo (tgo-IR), respectively, in control (upper) and KRASG12V (lower) third-instar larvae. Scale bar: 0.3 mm. (B) Quantification of total circulating hemocyte numbers with and without silencing of sima or tgo gene expression in control and KRASG12V third-instar larvae (n=6; results are presented as mean±s.d.; *P<0.05; Kruskal–Wallis H-test). (C) Left upper and lower panels show hemocytes (green) extracted from control and KRASG12V third-instar larvae, respectively, co-incubated with fluorescent Dextran-tagged S. aureus (red). Middle and right panels illustrate the effects of silencing HIF1A homolog sima (sima-IR) and ARNT homolog tgo (tgo-IR), respectively. Scale bar: 10 μm. (D) Quantification of phagosome area with and without silencing of sima or tgo gene expression in control and KRASG12V hemocytes (n=100; results are presented as mean±s.d.; *P<0.05; Kruskal–Wallis H-test). (E) Survival during development of control, KRASG12V and KRASG12V flies in which sima (sima-IR) or tgo (tgo-IR) were silenced in hemocytes. (F) Quantitative RT-PCR showing the relative expression level of sima and tgo in hemocytes from control compared to KRASG12V flies (n=3 replicates in each group; results are presented as mean±s.d.; *P<0.05; unpaired Student's t-test).
Fig. 6.
Fig. 6.
Echinomycin reverses KRASG12V-induced hemocyte overproliferation and immune deficiency. (A) Schematic illustration of the approach used for the KRASG12V Drosophila leukemia model drug screen (see Materials and Methods section for a detailed description of rationale, strategy and procedure). (B) Left upper and lower panels show control (Hml-Gal4, UAS-GFP) and KRASG12V (Hml-Gal4, UAS-GFP; UAS-KRASG12V) third-instar larvae, respectively, in which all hemocytes express GFP (green fluorescence). Middle and right panels show the effects of treating larvae with echinomycin and imatinib, respectively. Scale bar: 0.3 mm. (C) Quantification of total circulating hemocyte numbers without (vehicle) or with echinomycin or imatinib treatment in control and KRASG12V third-instar larvae (n=6; results are presented as mean±s.d.; *P<0.05; Kruskal–Wallis H-test). (D) Left upper and lower panels show hemocytes (green) extracted from control and KRASG12V third-instar larvae, respectively, co-incubated with fluorescent Dextran-tagged S. aureus (red). Middle and right panels illustrate the effects of treatment with echinomycin and imatinib, respectively. Scale bar: 10 μm. (E) Quantification of phagosome area in control and KRASG12V hemocytes with vehicle, echinomycin or imatinib (n=100; results are presented as mean±s.d.; *P<0.05; Kruskal–Wallis H-test).
Fig. 7.
Fig. 7.
Echinomycin reduces human leukemia cell viability and proliferation. (A) Cell viability of human leukemia cell lines following echinomycin treatment. Human leukemia cell lines were treated with echinomycin for 48 h at the indicated concentrations. Cell viability was then determined by incubating cells overnight with MTT tetrazolium salt (Sigma-Aldrich). Experiments were performed in triplicate. Red, leukemia cell lines carrying RAS mutations; green, leukemia cell lines in which RAS is not mutated. (B) Schematic illustration of THP1 cell (human leukemia cell line carrying NRASG12D) intrahepatic transplantation to generate mouse xenograft models, and time course of echinomycin treatments and diagnostic bioluminescence imaging to monitor THP1 cell transplant growth. (C) Bioluminescence imaging of mice engrafted with human THP1 leukemia cells bearing NRASG12D mutation. Mice were treated every other day with 10 μg/kg or 30 μg/kg echinomycin (as indicated) or vehicle control and were imaged after every third dose.

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