Actinomycin D Targets NPM1c-Primed Mitochondria to Restore PML-Driven Senescence in AML Therapy

Abstract

Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of promyelocytic leukemia (PML) nuclear bodies (NB), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mitochondrial DNA, activating cyclic GMP-AMP synthase signaling, and boosting reactive oxygen species (ROS) production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies.

Significance: ActD induces complete remissions in NPM1-mutant AMLs. We found that NPM1c affects mitochondrial biogenesis and PML NBs. ActD targets mitochondria, yielding ROS which enforce PML NB biogenesis and restore senescence. Dual targeting of mitochondria with ActD and venetoclax sharply potentiates their anti-AML activities in vivo. This article is highlighted in the In This Issue feature, p. 2945.

Figure 1.

NPM1c alters PML NBs biogenesis. A, Quantification (left) and representative image (right) of PML NBs in HSCs purified from NPM1c-expressing knock-in mice. The results are expressed as the mean value ± SD (error bars) of n = 5 NPM1^+/+ and 12 NPM1^cA/+ mice. Unpaired t test; ***, P < 0.001. Scale bar, 3 μm. B, Quantification (left) and representative image (right) of PML NBs in MEFs stably expressing GFP-PML-III and transiently transfected with NPM1-derived expression vectors. Results are expressed as the mean value ± SD (error bars) of n = 8 analyzed cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. C and D, Coimmunoprecipitation analyses of 293T cells transfected with indicated constructs. E, Histidine pulldown of disulfide-linked NPM1c–PML complex from transiently transfected 293T cells. *, nonspecific binding to NPM1. F, Quantification (left) and representative image (right) of PML NBs in isogenic AML2 derivatives. Results are expressed as the mean value ± SD (error bars) of n = 18 analyzed cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. G, Colony-forming ability of isogenic AML2 derivatives. Unpaired t test. n = 3. H, GSEA analyses of E2F and MYC pathways in transcriptomic exploration of NPM1c versus NPM1-expressing AML2 cells. I, Western blot analyses of TP53, ARF, PML, and endogenous NPM1 in isogenic AML2 cells. Quantification of WT NPM1 is shown. J, Western blot analyses of PML in primary bone marrow samples from 9 NPM1c and 7 NPM1 AML samples explored.

Figure 2.

NPM1c affects mitochondria and drives an integrated stress response. A, Number and volume of mitochondria in HSCs purified from NPM1c-knockin mice. The results are expressed as the mean value ± SD (error bars) of n = 8 NPM1^+/+ and 11 NPM1^cA/+ HSC. Unpaired t test; **, P < 0.005. Scale bar, 3 μm. B, Examination of mitochondrial morphology in NPM1-, NPM1c-, and NPM1c^C288S-expressing AML2 cells. Top, immunofluorescence analyses of mitochondrial morphology. Bottom, mitochondrial fragmentation was quantified by number of mitochondria, of junctions and branch length. The results are expressed as the mean value ± SD (error bars) of n = 25 cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. C, Transmission electron microscopy image of mitochondrial cristae in AML2-derived isogenic cells. Scale bar, 1 μm. D, FACS analyses of mitochondrial status in AML2-derived isogenic lines. The results from are expressed as the mean value ± SD of three independent experiments. Unpaired t test; ***, P < 0.001; **, P < 0.01. E, Quantification of cytosolic and mitochondrial mtDNA. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; ***, P < 0.001; n = 3. F, Immunofluorescence analyses of cGAS localization in NPM1c cells. G, Basal 2′3-cGAMP concentration in AML2 cells expressing NPM1c or not. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***, P < 0.001. n = 2. H, GSEA of differentially expressed genes in AML2 cells expressing NPM1c or not. I, Effect of NPM1c expression on nascent protein synthesis. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; ***, P < 0.001; n = 3. J, ATP/ADP ratio in AML2-derived isogenic lines. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; *, P < 0.05. K, Western blot analyses of TFAM and PGC1α in AML2-derived isogenic cells. Quantification of acetylated PGC1a is shown. L, Acetylation of PGC1α in isogenic AML2 cells. Immunoprecipitation of lysine-acetylated proteins followed by Western blot with an anti-PGC1α antibody. The estimated fraction of acetylated PGC1α is indicated.

Figure 3.

ActD targets mitochondria. A, Top, representative immunofluorescence analyses of mitochondrial morphology in AML3 cells treated with 5 nmol/L ActD. Bottom, mitochondrial fragmentation was quantified by number of mitochondria, of junction and branch length. The results are expressed as the mean value ± SD (error bars) of n = 25 cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 2 μm. B, Heat map (induction over mock-treated in the pool of samples) showing the expression of superoxide response genes upon ActD exposure. C and D, FACS analyses of mitochondrial membrane potential (C), ROS and mitochondrial superoxide production (D), after ActD exposure in AML2-derived isogenic cells. The results are expressed as the mean value ± SD. Unpaired t test. **, P < 0.005; ***, P < 0.001; n = 3. E, Effect of ActD on mtDNA abundance in the cytoplasm (left) or within mitochondria (right). Representative experiment of n = 2. F, Southern blot analysis of the different forms of mitochondrial DNA, in AML2 or AML3 cells treated or not with ActD (5 nmol/L) for 1 hour. G, levels of cytoplasmic cGAMP before or after ActD treatment. Representative experiment of n = 2. H, Western blot analyses of eIF2α and its phosphorylated form after ActD exposure. I, ATP/ADP ratio from metabolomic analyses of AML2 cell lines after ActD exposure. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***, P < 0.001. J, Western blot analyses of AMPK and its phosphorylated form after ActD exposure.

Figure 4.

ActD activates a ROS/PML/TP53 senescence axis ex vivo. A, Quantification (left) and representative image (right) of PML NBs upon ActD exposure in MEFs stably expressing GFP-PML-III and transiently expressing NPM1 (WT) or NPM1c (C+). The results are expressed as the mean value ± SD (error bars) of n = 10 cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. B,Ex vivo ActD treatment of primary AML blasts demonstrates PML NB restoration in NPM1c-AMLs. Representative images (left) and quantification (right). C, Histidine pulldown of disulfide-linked NPM1c–PML complex in transiently transfected 293T cells, before or after ActD exposure. D, Western blot analyses of AML3 and AML3^PML−/− cells after ActD treatment. E, SA-β-gal staining of AML3 derivatives 7 days after 2 hours ActD pretreatment. F, Effect of ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Representative experiment of n = 2. Unpaired t test; ***, P < 0.001. G, Western blot analyses of NAC pretreated AML cells upon ActD exposure. H, Effect of NAC and ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; ***, P < 0.001. Representative experiment of n = 2. I, Western blot analyses of ActD response of parental and mitochondria-depleted AML2 and AML3 cells. MT-CO1, Mitochondrial cytochrome c oxidase subunit 1; RXR, retinoid X receptor alpha.

Figure 5.

ActD exerts AML-specific growth suppression in vivo. A, Top, TP53 or HDM2 stabilization in bone marrow of immunodeficient mice xenografted with primary patient NPM1c-AML cells. +, Ex vivo MLL/ENL ActD-treated cells for 3 hours, as a positive control. Bottom, AML features in this mouse model following treated or not with ActD for 5 days. Unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Representative experiment of n = 3. B, Top, Trp53 stabilization in CD45.2⁺ NPM1c-driven murine AML blasts versus CD45.2⁻ normal mouse cells upon ActD exposure. Bottom, AML features after a week of ActD therapy. The results are expressed as the mean value ± SD. Unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Representative experiment of n = 3. C, Western blot analyses of AML3 and AML3^PML−/− engrafted immunodeficient mice treated or untreated with ActD for a week (left) and percentage of CD45⁺ AML3 blasts in the bone marrow (right). Unpaired t test. ***, P < 0.001. n = 2. D, Top, immunofluorescence analyses of blast-rich blood samples from an AML patient during ActD therapy. Bottom, quantification of PML NBs. Scale bar, 10 μm. E, Heat map of upregulated TP53 target genes in AML cells from ActD-treated patient. F, Western blot analyses of AML-rich blood samples during the first cycle of ActD therapy. G, GSEAs of differentially expressed genes in the patient treated with ActD. H, Expression of NFκB targets (left), acute stress response (middle), and proliferation genes (right) in AML-rich peripheral blood from patient during therapy (data from the microarray experiments).

Figure 6.

ActD enhances Venetoclax antileukemic effects. A, Mitochondrial morphology in AML3 cells treated with 5 nmol/L ActD and/or 1 μmol/L venetoclax for 3 hours. Left, mitochondrial fragmentation was quantified by number of mitochondria, of junction and branch length. Right, immunofluorescence analysis of mitochondrial morphology. The results are expressed as the mean value ± SD of n = 30 cells per condition. Unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.001. Scale bar, 5 μm. B–D, FACS analyses of general ROS (B), mitochondrial membrane potential (C), and mitochondrial superoxide production (D), after ActD and/or venetoclax exposure in AML3 cells. Cell were treated with indicated drugs for 3 hours in B and D and 6 hours in C. The results are expressed as the mean value ± SD from from n = 2 experiments. Unpaired t test. ***, P < 0.001. E, Leakage of mitochondrial DNA to the cytoplasm in response to a 3-hour ActD or venetoclax exposure in AML3 cells. Results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***, P < 0.001. Representative experiment of n = 2. F, 2′3-cGAMP concentration in AML cells after 6 hours exposure to venetoclax and/or ActD. Representative experiment of n = 2. G, Methylcellulose colony formation assays of AML cell lines upon 2 hours preseeding exposure to venetoclax and/or ActD. Results are expressed as the mean value of triplicate samples ± SD. Unpaired t test. ***, P < 0.001. Representative experiment of n = 3. H, Survival of immunodeficient mice xenografted with AML3 or AML3^PML−/− cells and treated with ActD or venetoclax, 5 days per week. I, AML features in NPM1c-driven AML mice treated with ActD and or not venetoclax for 5 days. The results are expressed as the mean value ± SD. Unpaired t test. *, P < 0.05; ***, P < 0.001. n = 2. J, Response to ActD and/or venetoclax in a transplantable AML model initiated by NPM1c + IDH1^R132H mutation. Left, GFP abundance in bone marrow samples collected at treatment interruption (arrow in the survival curve, right), Representative experiment of n = 3. K, Abundance of human AML cells in xenografted mice treated for two weeks. Combined therapy induces AML regression. L, Model of venetoclax/ActD/mitochondria/PML/TP53 interplays in NPM1c-AMLs. Dashed lines point to likely mechanisms not directly explored here.