Abstract

   Pyrvinium pamoate, a widely-used anthelmintic agent, reportedly
   exhibits significant anti-tumor effects in several cancers. However,
   the efficacy and mechanisms of pyrvinium against myeloid leukemia
   remain unclear. The growth inhibitory effects of pyrvinium were tested
   in human AML cell lines. Transcriptome analysis of Molm13 myeloid
   leukemia cells suggested that pyrvinium pamoate could trigger an
   unfolded protein response (UPR)-like pathway, including responses to
   extracellular stimulus [p-value = 2.78 × 10^−6] and to endoplasmic
   reticulum stress [p-value = 8.67 × 10^−7], as well as elicit metabolic
   reprogramming, including sulfur compound catabolic processes [p-value =
   2.58 × 10^−8], and responses to a redox state [p-value = 5.80 × 10^−5];
   on the other hand, it could elicit a pyrvinium blunted protein folding
   function, including protein folding [p-value = 2.10 × 10^−8] and an ATP
   metabolic process [p-value = 3.95 × 10^−4]. Subsequently, pyrvinium was
   verified to induce an integrated stress response (ISR), demonstrated by
   activation of the eIF2α-ATF4 pathway and inhibition of mTORC1
   signaling, in a dose- and time-dependent manner. Additionally,
   pyrvinium could co-localize with mitochondria and then decrease the
   mitochondrial basal oxidative consumption rate, ultimately
   dysregulating the mitochondrial function. Similar effects were observed
   in cabozantinib-resistant Molm13-XR cell lines. Furthermore, pyrvinium
   treatment retarded Molm13 and Molm13-XR xenograft tumor growth. Thus,
   we concluded that pyrvinium exerts anti-tumor activity, at least, via
   the modulation of the mitochondrial function and by triggering ISR.

   Keywords: acute myeloid leukemia, FLT3-ITD, pyrvinium pamoate,
   integrated stress response

1. Introduction

   Acute myeloid leukemia (AML) is a heterogeneous hematological
   malignancy, characterized by the abnormal proliferation and
   differentiation blockage of progenitor stem cells, leading to
   leukemogenesis [[38]1]. The past decades have witnessed substantial
   progress in the biological classification of AML [[39]2]. Drug therapy
   for AML underwent significant advances in 2017 and 2018, with the US
   Food and Drug Administration (FDA) approving several new targeted
   agents [[40]3,[41]4]. Among these, midostaurin [[42]5] and gilteritinib
   [[43]6] were reported to be suitable for AML patients with a fms-like
   tyrosine kinase 3 (FLT3) mutation. The use of these specific FLT3
   inhibitors alone exhibited relatively high response rates (50–66%);
   however, the duration of responses and survival were found to be brief,
   with patients living just 20–33 weeks [[44]7]. Therefore, the
   development of more effective and long-lasting treatment strategies is
   warranted, especially for AML patients with the FLT3 mutation, which is
   among the most common mutations in AML [[45]8]. In recent years,
   researchers have attempted to use established, FDA-approved drugs, to
   help devise appropriate treatment options.

   Pyrvinium pamoate is an FDA-approved anthelminthic drug used for the
   treatment of pinworms in humans [[46]9]. The treatment of pinworm
   infections in humans (enterobiasis) with pyrvinium pamoate was reported
   as early as 1959 [[47]10]. Recent studies have shown that pyrvinium
   selectively inhibits the growth of tumor cells of diverse tissue
   origins, including pancreas, colon, breast, brain, myeloma, and other
   hematological malignancies, without affecting the normal, healthy cells
   of the body [[48]11,[49]12,[50]13]. The mechanism of the action of
   pyrvinium appears to vary in different tumor types. The following
   mechanisms have been elucidated: (1) through the allosteric activation
   of casein kinase 1α and the destabilization of β-catenin in
   wingless-related integration site (Wnt) pathways [[51]14,[52]15], as
   well as related Hedgehog signaling pathways [[53]16]; (2) through the
   inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B
   (AKT) signaling pathway [[54]17]; (3) through the suppression of
   mitochondrial respiratory complex I or II [[55]12,[56]18,[57]19], and
   the regulation of signal transducer and activator of transcription 3
   (STAT3) activation [[58]12]; (4) by targeting the unfolded protein
   response (UPR) during hypoglycemia [[59]20]; (5) by targeting autophagy
   addiction [[60]21]; and (6) by acting as a non-competitive androgen
   receptor inhibitor [[61]22,[62]23]. Of note, several studies exploring
   drug-repurposing have also reported pyrvinium as a potential
   anti-cancer compound [[63]17,[64]24,[65]25,[66]26,[67]27,[68]28].
   However, the anti-cancer effect of pyrvinium on AML, and the underlying
   mechanisms, remain unclear.

   Therefore, this study aimed to evaluate the in vitro and in vivo
   effects of pyrvinium in Molm13 leukemia cells, and conduct
   transcriptomic analysis to elucidate the mechanism through which it
   acts. Additionally, we also evaluated the effects of pyrvinium against
   a cabozantinib-resistant Molm13 cell line (Molm-13-XR).

2. Materials and Methods

2.1. Cell Culture and Chemicals

   The human AML cell lines Molm13 (DSMZ, Brauschweig, Germany) and MV4-11
   (DSMZ) were maintained in Roswell Park Memorial Institute (RPMI)-1640
   medium (ThermoFisher Scientific/Gibco, Waltham, MA, USA), containing
   10% fetal bovine serum (FBS); Kasumi-1 cells (ATCC, Manassas, VA, USA)
   were cultured in RPMI-1640 medium containing 20% FBS. The
   cabozantinib-resistant cell line, Molm13-XR, was established by
   culturing Molm13 cells in the presence of increasing concentrations of
   cabozantinib. The stable resistant cell line was then maintained in a
   complete RPMI-1640 medium with 10% FBS, in the absence of cabozantinib.
   Cell lines were cultured at 37 °C in a humidified atmosphere containing
   5% CO[2]. The genetic profiles of these cell lines were authenticated
   (16-markers short tandem repeat) by the Food Industry Research and
   Development Institute (Hsinchu, Taiwan), and were found to be identical
   to their reported generic profiles.

   Cabozantinib-malate, pyrvinium pamoate, and rapamycin were purchased
   from Selleck Chemicals (Houston, TX, USA), Sigma-Aldrich/Merck
   (Darmstadt, Germany), and TargetMol (Boston, MA, USA), respectively.

2.2. Cell Viability

   The cells were exposed to the indicated doses of various compounds for
   72 h, and the cell viability was measured using the Cell Titer 96
   AQueous One Solution Cell Proliferation Assay (MTS assay; Promega,
   Madison, WI, USA), as per the manufacturer’s instructions. The half
   maximal inhibitory concentration (IC[50]) was determined using the
   CalcuSyn software (ver. 2.11; Biosoft, Cambridge, UK).

2.3. Cell Cycle Analysis

   After incubating for 24 or 72 h with the drugs, the cells were
   harvested and washed with ice-cold phosphate buffered saline (PBS). The
   washed cells were then added to ethanol (Sigma-Aldrich/Merck,
   Darmstadt, Germany) while slowly vortexing, and were frozen at −20 °C
   for at least 24 h. The fixed cells were washed twice with PBS, stained
   with propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) for
   15 min and analyzed using a CytoFLEX flow cytometer (Beckman Coulter,
   Brea, CA, USA).

2.4. Immunoblotting

   As previously described [[69]29], protein lysates were separated in 10%
   polyacrylamide gel, and transferred to a polyvinylidene fluoride
   membrane. After blocking the non-specific binding sites, membranes were
   incubated with primary antibodies ([70]Supplementary Table S1) at 4 °C
   overnight. Subsequently, membranes were washed three times with
   Tris-buffered saline containing polysorbate 20 (TWEEN 20) and incubated
   with horseradish peroxide-conjugated secondary antibodies (Cell
   Signaling Technology, Beverly, MA, USA) at room temperature for 1 h.
   Chemiluminescence signals were then generated by Western Lightening
   Plus-ECL (Perkin Elmer, Waltham, MA, USA) and captured by LAS4000
   (Fujifilm, Tokyo, Japan). Quantification of protein and phosphorylation
   levels was performed by ImageJ (1.51 K).

2.5. Transcriptomic Analysis of Pyrvinium-Treated Molm13 Cells

   Total RNA was isolated from the cells using the NucleoSpin^® RNA kit
   (Macherey-Nagel, Düren, Germany), after treatment with dimethyl
   sulfoxide (DMSO) or 100 nM pyrvinium for 6 h. The viability of the
   Molm13 cells collected for RNA extraction was more than 90% after this
   treatment. The RNA (1 μg) was then used for library preparation. A
   strand-specific RNA library was built using TruSeq stranded mRNA
   library prep Kit (cat# RS-122-2101, Illumina, San Diego, CA, USA), as
   per the manufacturer’s protocol. The libraries were then sequenced
   using the Illumina NovaSeq 6000 platform and 75 bps paired end reads
   were generated (Genomics, Taipei, Taiwan).

   Sequencing alignment with hg19 was then performed using Rsubread
   (Bioconductor) [[71]30], and differentially expressed genes (DEGs)
   analysis in the DMSO and pyrvinium groups was performed using EBSeq
   (Bioconductor) [[72]31]. A threshold of ≥2-fold (or ≤0.5-fold) and
   p-value of ≤0.05 were set and used to screen DEGs. The transcriptome
   files can be found in the Gene Expression Omnibus (GEO) database (ID:
   [73]GSE153854). Pathway enrichment was then analyzed by gene set
   enrichment analysis (GSEA) [[74]32] using the Molecular Signatures
   Database v7.0 (MSigDB, Broad Institute, Cambridge, MA, USA) and
   Metascape [[75]33]. The expression of genes was validated by
   quantitative reverse transcription-PCR (q-RT-PCR), according to methods
   previously described [[76]29]. The primers used for specific gene
   detection are listed in [77]Supplementary Table S2.

2.6. Confocal Microscopy Observation

   After drug exposure for 24 h, PBS-washed cells were re-suspended in
   serum-free RPMI 1640 medium containing 100 μM MitoTracker Green
   (ThermoFisher Scientific/Gibco) and incubated for 30 min in a culture
   incubator. Mitochondria-stained cells were centrifuged again and
   stained with 3 μM of 4,6-diamidino-2-phenylindole (DAPI) for 15 min.
   Finally, the cells were washed again with serum-free medium and
   re-suspended in PBS. Cell suspensions were dropped on a glass slide and
   observed by confocal microscopy. The images of Confocal Microscopy were
   acquired by Zeiss LSM 880 and analyzed by ZEN software (Carl ZEISS,
   Jena, Germany).

2.7. Cellular Bioenergetic Analysis

   The oxygen consumption rate (OCR) and extracellular acidification rate
   (ECAR) were analyzed in real-time using the XFe24 extracellular flux
   analyzer (Agilent Technologies/Seahorse Bioscience, Billerica, MA,
   USA), as per the manufacturer’s protocol. After the indicated drug
   treatment for 24 h, cells were seeded in the wells (1 × 10^5
   cells/well) of a 24-well microplate, precoated with Cell-Tak Cell and
   Tissue Adhesive (Corning, Corning, NY, USA). The Cell Mito Stress Test
   kit (Agilent Technologies/Seahorse Bioscience) was used for the
   measurement of mitochondrial respiration. After determination of the
   baseline respiration, oligomycin A, carbonyl-cyanide
   p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and antimycin A were
   sequentially applied on the microplate to determine adenosine
   triphosphate (ATP)-linked respiration, protein leak, maximal
   respiratory capacity, and reserve capacity. The Glycolysis Stress Test
   kit (Agilent Technologies/Seahorse Bioscience) was used to assess the
   glycolytic activity in the cells following a sequential addition of
   glucose, oligomycin, and 2-deoxglucose (2-DG) in order to examine
   glycolysis, glycolytic capacity, and glycolytic reserve. The results
   were analyzed by Seahorse XFe24 wave software 2.2 (Agilent
   Technologies/Seahorse Bioscience).

2.8. Mitochondrial Parameters (Mitochondrial Mass and Reactive Oxygen Species
(ROS) Levels)

   The mitochondrial mass and ROS levels in cells were measured by flow
   cytometer (CytoFLEX flow cytometer, Beckman Coulter) using MitoTracker
   Green FM (Invitrogen/ThermoFischer Scientific, Waltham, MA, USA) and
   dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich, St.
   Louis, MI, USA), respectively. To evaluate the mitochondrial mass in
   the living cells, drug-treated cells (1 × 10^6) were re-suspended in
   RPMI -1640 serum-free medium containing 100 nM MitoTracker Green and
   incubated for 30 min under growth conditions. To determine the ROS
   levels, cells were stained with 10 μM DCFH-DA for 30 min at 37 °C in
   the dark, washed, and re-suspended in PBS.

2.9. Mitochondrial Complex I Activity

   The mitochondria complex I activity was determined using the complex I
   enzyme activity microplate assay kit (Abcam, Cambridge, UK), as per the
   manufacturer’s instruction. The cells were exposed to the drugs for 24
   h before being harvested, washed, and re-suspended in PBS. The protein
   concentration of the pellets was determined using the Bradford protein
   assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The pellets were
   diluted to 5.5 mg/mL with PBS. Cells were then lysed with 10× detergent
   provided in the kit and diluted with incubation solution. Finally, 200
   μg protein lysate was added to the 96-well microplate and incubated for
   3 h. It was then washed thrice with a buffer solution. After the assay
   solution was added, A[450] absorbance signals were detected kinetically
   every 20 s for 30 min using a Spectra Max M5 microplate reader
   (Molecular Devices).

2.10. Subcutaneous Mouse Xenografts

   To assess the efficacy of pyrvinium pamoate in vivo, a mouse
   subcutaneous xenograft model was established in Female
   CAnN.Cg-Foxn1^nu/CrlNarl (nude) mice. Nude mice aged 6–8 weeks were
   purchased from the National Laboratory Animal Center, NARLabs, Taiwan.
   The mice were anesthetized by inhaling isoflurane and maintained with
   2% isoflurane, followed by subcutaneous inoculation in the flank with 9
   × 10^6 Molm13 or Molm13-XR cells suspended in a 1:1 homogeneous mixture
   of PBS and Matrigel Matrix (Corning). The tumor volume was calculated
   as follows: volume (mm^3) = ½ × (width)^2 × length. When the tumor size
   reached 200 mm^3, mice were randomly divided into three groups (n =
   8–10 mice/group), and DMSO or pyrvinium (0.5 or 0.8 mg/kg in 20% DMSO
   in PBS) was administered for six days, followed by a one-day rest. The
   protocol for the xenograft experiments was approved by the
   Institutional Animal Care and Use Committee (IACUC) of the College of
   Medicine, National Taiwan University (no. 20170161, 19 July 2017), and
   conformed with the criteria outlined in the Guide for the Care and Use
   of Laboratory Animals prepared by the National Academy of Sciences and
   published by the National Institutes of Health.

2.11. Statistical Analysis

   The differences between the measured values were evaluated using a
   two-sided Student’s t-test. The error bars in the graphs indicate the
   means ± standard deviation, or means ± standard error of means of at
   least three independent experiments, conducted in triplicate. Values of
   p < 0.05 were considered statistically significant (* p < 0.05; ** p <
   0.01; *** p < 0.001).

3. Results

3.1. Pyrvinium Pamoate Can Inhibit Cell Proliferation and Induce Cell Death
in Molm13 Cells

   To evaluate the efficacy of pyrvinium against leukemia, several myeloid
   leukemia cell lines were examined. Among them, FLT3-ITD-harboring
   leukemic cells Molm13 were the most sensitive to pyrvinium treatment,
   exhibiting an IC[50] of 50.15 ± 0.43 nM ([78]Figure 1A). Flow cytometry
   analysis showed that pyrvinium triggered cell cycle arrest in the
   G[0]/G[1] phase and increased the sub-G[1] population ([79]Figure 1B).
   Immunoblotting also revealed the decreased levels of cyclin E1 protein
   following pyrvinium treatment ([80]Figure 1C). In brief, pyrvinium
   induced cell cycle arrest and apoptosis-mediated cytotoxicity at doses
   in the nanomolar range. In this study, we used the percentage of
   sub-G[1] populations instead of the percentage of PI/Annexin V (+)
   populations to evaluate the extent of apoptosis, since the fluorescence
   background interference of pyrvinium in the PI/Annexin V signals in
   flow cytometry may have compromised the reliability of the results
   [[81]34]. The immunoblotting results revealed that FLT3 downstream
   signaling, including AKT, STAT5, and ERK, in Molm13 cells was only
   slightly inhibited by pyrvinium ([82]Figure 1D). Further examination
   showed that pyrvinium did not inhibit other pathways known to be
   targeted by pyrvinium in other types of tumors, such as the STAT3 and
   Wnt pathways ([83]Figure 1D,E); these results suggested that
   alternative mechanisms, including but not limited to mitochondrial
   mechanisms, may contribute to this robust anti-leukemic activity of
   pyrvinium.

Figure 1.

   [84]Figure 1
   [85]Open in a new tab

   In vitro effect of pyrvinium pamoate on FLT3-ITD harboring Molm13
   myeloid leukemia cells. (A) Half-maximal inhibitory concentration of
   pyrvinium after 72 h of treatment in Molm13, MV4-11, and Kasumi-1
   cells, evaluated by MTS assay. (B) After exposure to pyrvinium for 72
   h, the different populations of Molm13 cells were analyzed by flow
   cytometry using propidium iodide staining. * p < 0.05, ** p < 0.01, ***
   p < 0.001 vs. DMSO. (C) Immunoblot analysis of indicated proteins in
   Molm13 cells treated with DMSO, 50 nM pyrvinium, and 100 nM pyrvinium
   for 24 h and probed with anti-cyclin E1 antibody. (D) Immunoblot
   analysis of indicated proteins in Molm13 cells exposed to drugs for 4
   h; or (E) probed with anti-p-GSK3β, GSK3β, p-β-catenin, and β-catenin
   antibodies. α-Tubulin was used as a loading control. Representative
   Western blots of three independent experiments are shown. Values
   represent the fold change with respect to the DMSO control group, after
   normalization with α-tubulin or β-actin.

3.2. Transcriptome Analysis Revealed That Pyrvinium Pamoate Triggers an
UPR-like Pathway and Redox Balance Modulation, but Blunts Protein Folding
Function and ATP Synthesis in Molm13 Cells

   To elucidate the alternative mechanisms involved in the action of
   pyrvinium in Molm13 leukemic cells, we performed whole transcriptome
   analysis (RNA-seq) on Molm13 cells after treatment with 100 nM
   pyrvinium for 6 h. A total of 219 genes were identified as significant
   DEGs between the pyrvinium-treated and the control group, at p-value ≤
   0.05. Gene expression was considered to be significantly up- or
   down-regulated when [log[2] (fold change)] ≥ 1 or ≤−1, respectively.
   Among them, 162 upregulated and 57 downregulated genes were observed in
   the group treated with pyrvinium. Pathway enrichment analysis conducted
   by Metascape showed that genes upregulated after pyrvinium treatment
   were strongly associated with ‘response to extracellular stimulus’
   [p-value = 2.78 × 10^−6, q-value = 0.0079], ‘endoplasmic reticulum
   stress’ [p-value = 8.67 × 10^−7, q-value = 0.0039] or ‘redox balance’
   [p-value = 5.80 × 10^−5, q-value = 0.0492]; as well as with pathways
   associated with ‘sulfur compound catabolic process’ [p-value = 2.58 ×
   10^−8, q-value = 0.0003] and ‘amino acid transport’ [p-value = 1.02 ×
   10^−6, q-value = 0.0039] ([86]Figure 2A). A volcano plot was used to
   depict the genes associated with responses to extracellular stimulus
   (such as, SESN2, and PCK2), redox balance (CTH and CHAC1) and amino
   acid metabolism (ASNS, GPT2, PSAT1, SLC7A11, and SLC3A2) ([87]Figure
   2C; [88]Supplementary Table S3). GSEA of the transcriptome ([89]Figure
   2D, upper) indicated a trend consistent with that of a documented gene
   signature changed after amino acid deprivation [[90]35].

Figure 2.

   [91]Figure 2
   [92]Open in a new tab

   Transcriptomic analysis of 100 nM pyrvinium-treated Molm13 cells. (A)
   Pathway enrichment analysis of upregulated differentially expressed
   genes (DEGs) in Molm13 cells exposed to 100 nM pyrvinium for 6 h;
   analysis was performed using Metascape. (B) Volcano plot depicting the
   DEGs upregulated or downregulated by treatment with 100 nM pyrvinium.
   Orange triangle: ATF4 downstream genes; pink diamond: heat shock
   protein genes; green dot: mitochondria-encoded genes. (C) Gene set
   enrichment analysist plot of Molm13 cells treated with 100 nM pyrvinium
   showing the upregulation of genes associated with amino acid
   deprivation (upper), and the inhibition of HSF1 activation (lower).
   NES: normalized enrichment score. (D) Pathway enrichment analysis of
   downregulated DEGs in Molm13 cells exposed to 100 nM pyrvinium for 6 h;
   analysis was performed using Metascape. (E) Schematic picture of
   potential metabolic gene triggered after pyrvinium treatment in Molm13
   cells.

   In contrast, the genes that were differentially downregulated after
   pyrvinium treatment were enriched in the pathway associated with
   ‘protein folding modulation’ (response to topologically incorrect
   protein) [p-value = 2.10 × 10^−8, q-value = 0.0005], including several
   heat shock proteins (HSPs), such as HSP90B1 (encodes GRP94, p = 0.053),
   HSPA5 (encodes GRP78), HSPA8, HSPH1, and DNAJA1 (HSPF4), and those
   associated with ‘ATP synthesis’ (ATP metabolism) [p-value = 3.95 ×
   10^−4, q-value = 0.1736], including MT-ND5 and MT-COX3 ([93]Figure
   2B,C; [94]Supplementary Table S3). GSEA showed that the changes in the
   transcriptome profile induced by pyrvinium were opposite to the changes
   in genes after heat shock factor 1 (HSF1) activation ([95]Figure 2D,
   lower). Based on the results of these transcriptomic analyses, we
   proposed that pyrvinium could induce a stress response, causing an
   amino acid deprivation-like scenario, and elicit metabolic rewiring
   toward amino acid synthesis, transport, and glutathione metabolism
   ([96]Figure 2E).

3.3. Pyrvinium Pamoate Targets Mitochondria and Has an Inhibitory Effect on
Mitochondrial Respiration

   Pyrvinium has been reported to co-localize with mitochondria and cause
   mitochondrial stress, leading to the inhibition of complex I activity,
   impediment of mitochondrial oxidative phosphorylation (OXPHOS), and a
   decreased ATP production in human myeloma and erythroleukemia cell
   lines [[97]12]. To clarify whether treatment with pyrvinium could alter
   the mitochondria function in Molm13 leukemia cells, the cellular
   location of pyrvinium, electron transport chain enzyme activity
   (especially complex I), and ATP production were examined. MitoTracker
   Green staining, coupled with confocal microscopy, showed that the red
   fluorescence of pyrvinium overlapped with the green fluorescence of the
   mitochondria, indicating that pyrvinium was localized in the
   mitochondria ([98]Figure 3A). Subsequently, Seahorse bioanalyzer
   analysis revealed that pyrvinium had an inhibitory effect on the
   mitochondrial basal respiration rate, spare respiratory capacity,
   proton leak, and ATP production after treatment with 10 nM pyrvinium
   for 24 h ([99]Figure 3B). Further evaluation of the ROS levels and
   total mitochondrial mass was conducted by DCFH-DA and MitoTracker Green
   staining, respectively. This showed that pyrvinium increased ROS levels
   ([100]Figure 3C), but reduced the mass of mitochondria ([101]Figure
   3D). Finally, the dose-dependent inhibition of the mitochondrial
   complex I was noted ([102]Figure 3E). On the other hand, to evaluate
   the effect of pyrvinium on glycolytic activity in Molm 13 cells, the
   Glycolysis Stress Test was performed, revealing that basal glycolysis
   and glycolytic capacity were increased after pyrvinium treatment
   ([103]Figure 3F). In conclusion, pyrvinium could trigger mitochondrial
   stress by increasing ROS levels and impede mitochondrial functions, as
   indicated by the decreased OCR, ATP production, mitochondrial mass, and
   complex I activity.

Figure 3.

   [104]Figure 3
   [105]Open in a new tab

   Mitochondria functions were impaired upon exposure to pyrvinium. (A)
   Confocal microscopy images showing that pyrvinium co-localized with
   mitochondria in Molm13 cells. Arrows indicate the mitochondria
   overlapped with pyrvinium. (B) Seahorse Cell Mito Stress Test was
   performed to measure basal respiratory, spare respiratory capacity,
   proton leak, and ATP-linked respiration in Molm13 cells treated with 5
   nM, or 10 nM pyrvinium for 24 h compared to those in cells treated with
   DMSO. (C) Intracellular reactive oxygen species levels were measured by
   DCFH-DA staining; (D) Mitochondria mass was determined by MitoTracker
   Green staining coupled with flow cytometry in Molm13 cells treated with
   indicated concentrations of pyrvinium for 24 h. (E) Mitochondria
   respiratory chain complex I activity was measured after treatment with
   DMSO, 10 nM pyrvinium or 25 nM pyrvinium for 24 h. (F) Seahorse
   Glycolysis Stress Test was performed to measure basal glycolysis,
   glycolytic capacity, and glycolytic reserve after pyrvinium or DMSO
   treatment for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO.

3.4. Pyrvinium Pamoate Triggers the Activation of the eIF2α-ATF4 Pathway and
Inhibits mTORC1 Signaling

   To elucidate the role of integrated stress response (ISR) in the
   activity of pyrvinium, the phosphorylation of eukaryotic initiation
   factor 2α (eIF2α) and the expression of activating transcription factor
   4 (ATF4) were examined. A dose-dependent and time-dependent increase in
   the total ATF4 and phosphorylated elF2α levels was noted following
   pyrvinium treatment ([106]Figure 4A). In addition, the expression of
   various ATF4-regulated genes, which are associated with stress
   response, redox balance, and amino acid synthesis or transport, was
   also increased in a dose-dependent manner; this was confirmed by
   q-RT-PCR ([107]Figure 4B). These findings indicated that pyrvinium may
   elicit a cellular stress response via the eIF2α-ATF4 signaling pathway.
   Notably, the gene expression of TRIB3 (involved in C/EBP homologous
   protein-10 [CHOP]-related cell death) [[108]36], PMAIP1 (encoding
   proapoptotic NOXA), BBC3 (encoding proapoptotic p53 upregulated
   modulator of apoptosis [PUMA]), and DDIT3 (CHOP) was also stimulated
   after 24 h of pyrvinium treatment ([109]Figure 4B); this is in line
   with the pyrvinium-induced apoptosis which was observed in [110]Figure
   1B. Upregulation of the protein level of PUMA was also observed after
   exposure to pyrvinium for 24 h ([111]Figure 4C). In addition, increased
   gene expression of DNA damage inducible transcript 4 (DDIT4) and
   sestrin 2 (SESN2), two ATF4-regulated mTORC1 inhibitors, was noted
   following pyrvinium exposure ([112]Figure 4B). Western blotting also
   revealed that the phosphorylation of the mTORC1 downstream proteins,
   ribosomal protein S6 kinase 1 (S6K) and eukaryotic translation
   initiation factor 4E-binding protein 1 (4E-BP1), was reduced after 24 h
   treatment with pyrvinium ([113]Figure 4D); however, the phosphorylation
   of the protein kinase R-like endoplasmic reticulum kinase (PERK) and
   general control nonderepressible 2 (GCN2) was not obviously increased
   ([114]Figure 4E). Therefore, we suggested that pyrvinium could trigger
   the activation of the eIF2α-ATF4 pathway, in a PERK- and
   GCN2-independent manner, to promote the expression of its downstream
   effectors, DDIT4 and SESN2, and thus inhibit the mTORC1 signaling
   pathway. This is in line with the report that the eIF2α-ATF4 pathway
   could be activated through mitochondrial stress, without classical
   kinase activation [[115]37].

Figure 4.

   [116]Figure 4
   [117]Open in a new tab

   Pyrvinium modulated signaling pathways associated with the integrated
   stress response in Molm13 cells. (A) Western blot analysis of Molm13
   cells treated with DMSO, 50 nM pyrvinium, or 100 nM pyrvinium, then
   probed with antibodies against p-eIF2α, eIF2α, ATF4, and α-tubulin
   (loading control) for 6 h (left) and 24 h (right). (B) q-RT-PCR
   analyses of indicated genes in Molm13 cells upon exposure to DMSO, 50
   nM pyrvinium, or 100 nM pyrvinium for 24 h. * p < 0.05, ** p < 0.01,
   *** p < 0.001 vs. DMSO. Western blot analysis of Molm13 treated with
   DMSO, 50 nM pyrvinium, 100 nM pyrvinium, or 20 nM rapamycin (positive
   control), then probed with antibodies against (C) PUMA, (D) p-mTOR,
   mTOR, p-4EBP, 4EBP, p-S6K, or S6K, (E) p-GCN2, GCN2, p-PERK, or PERK,
   and α-tubulin (loading control) for 24 h. Representative Western blots
   of three independent experiments are shown. Values represent the fold
   change with respect to the DMSO control group after normalization with
   α-tubulin.

3.5. Pyrvinium Pamoate Exhibited Similar Activity in Molm13 Cells and
Cabozantinib-Resistant Molm13-XR Cells

   Cabozantinib is a tyrosine kinase inhibitor (TKI) that exhibits
   substantial activity against specific myeloid leukemia cell lines with
   FLT3-internal tandem duplication (ITD) [[118]38] or AML1-ETO/KITmt
   [[119]29] mutations, including MV4-11, Molm13, and Kasumi-1 cells. It
   is known that most patients with AML carrying FLT3-ITD show an initial
   favorable response to TKIs, such as midostaurin and gilteritinib;
   however, the emergence of drug-resistance can decrease drug efficacy
   [[120]4]. To investigate whether pyrvinium can act on TKI-resistant
   cell lines, we established a cabozantinib-resistant Molm13 cell line
   (Molm13-XR) by exposing Molm13 cells to gradually increasing
   concentrations of cabozantinib. The IC[50] of cabozantinib increased
   from 1.06 ± 0.93 nM in Molm13 cells to 473.36 ± 154.73 nM in Molm13-XR
   cells.

   We subsequently investigated the efficacy of pyrvinium against
   Molm13-XR cells. Similar to the results in Molm13 cells, pyrvinium also
   inhibited the proliferation of Molm13-XR in a dose-dependent manner,
   exhibiting an IC[50] of 115.5 ± 23.04 nM ([121]Figure 5A). Pyrvinium
   also induced apoptosis ([122]Figure 5B), decreased the population of
   cells in the G[2]/M phase, and decreased the cyclin E1 protein level in
   Molm13-XR cells after treatment for 72 h ([123]Figure 5C).
   Additionally, pyrvinium also inhibited the AKT, STAT5, and ERK
   signaling pathways ([124]Figure 5D). Pyrvinium also triggered eIF2α
   activation, increased ATF4 protein levels, and inhibited the mTOR
   signaling pathway ([125]Figure 5E). Pyrvinium could co-localize with
   mitochondria ([126]Figure 5F) and further reduce the mitochondria basal
   respiratory rate and mitochondrial ATP production ([127]Figure 5G) in
   the cabozantinib-resistant cell line. Similarly, pyrvinium could
   increase ROS levels, decrease mitochondrial mass, and inhibit
   mitochondria complex I activity ([128]Figure 5H–J) in Molm13-XR cells.
   On the other hand, the Glycolysis Stress Test revealed that only basal
   glycolysis was increased but glycolytic capacity was decreased after
   pyrvinium treatment ([129]Figure 5K).

Figure 5.

   [130]Figure 5
   [131]Open in a new tab

   In vitro anti-leukemic effect of pyrvinium in Molm13-XR cells. (A) Half
   maximal inhibitory concentration of pyrvinium after treatment for 72 h,
   evaluated by the MTS assay in Molm13, and Molm13-XR cells. (B)
   Different populations of Molm13-XR cells upon exposure to pyrvinium for
   72 h were evaluated by flow cytometry using propidium iodide staining.
   * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DMSO. (C) Western blot
   analysis of Molm13-XR cells treated with DMSO, 50 nM pyrvinium, or 100
   nM pyrvinium for 24 h and probed with an anti-cyclin E1 antibody. (D)
   Western blot analysis of indicated proteins in Molm13-XR cells exposed
   to drugs for 4 h. (E) Western blot analysis of Molm13-XR cell lysates
   probed with anti-p-eIF2α, eIF2α, and ATF4 antibodies for 6 h (E, upper
   left) or 24 h (E, lower left); or probed with anti-p-mTOR, mTOR,
   p-4EBP, 4EBP, p-S6K, S6K, p-GCN2, GCN2, p-PERK, PERK antibodies for 24
   h (E, middle and right). α-Tubulin was used as a loading control.
   Representative Western blots of three independent experiments are
   shown. Values represent the fold change with respect to the DMSO
   control group after normalization with α-tubulin. (F) Confocal
   microscopy images demonstrating that pyrvinium was co-localized with
   mitochondria in Molm13-XR cells. Arrows indicate the mitochondria
   overlapped with pyrvinium. (G) Seahorse Cell Mito Stress Test was
   performed to measure basal respiratory, spare respiratory capacity,
   proton leak, and the ATP-linked respiratory in Molm13-XR cells treated
   with 5 nM, or 10 nM pyrvinium for 24 h, compared with those in cells
   treated with DMSO. (H) Intracellular reactive oxygen species levels
   were measured by DCFH-DA staining; (I) Mitochondria mass was determined
   by MitoTracker Green staining coupled with flow cytometry in Molm13-XR
   cells treated with indicated concentrations of pyrvinium for 24 h. (J)
   Mitochondria respiratory chain complex I activity was measured after
   treatment with DMSO, 10 nM pyrvinium or 25 nM pyrvinium for 24 h. (K)
   Seahorse Glycolysis Stress Test was performed to measure basal
   glycolysis, glycolytic capacity, and glycolytic reserve after pyrvinium
   or DMSO treatment for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001 vs.
   DMSO.

3.6. Pyrvinium Pamoate Retards the Growth of Subcutaneous Molm13 and
Molm13-XR Xenograft Tumors

   To determine the antitumor efficiency of pyrvinium in vivo, mice
   bearing Molm13 and Molm13-XR tumors were injected intraperitoneally
   with pyrvinium (0.5 or 0.8 mg/kg), six days a week. We found that the
   pyrvinium treatment retarded Molm13 ([132]Figure 6A,B, left) and
   Molm13-XR ([133]Figure 6A,B, right) tumor growth in a dose-dependent
   manner. Compared with the control group (DMSO), intraperitoneal
   administration of pyrvinium at a dose of 0.8 mg/kg could significantly
   prolong the survival time of mice inoculated with Molm13 and Molm13-XR
   tumors ([134]Figure 6C). The drug was well-tolerated in mice and no
   effects on the body weight were observed during the dosing period
   ([135]Figure 6D).

Figure 6.

   [136]Figure 6
   [137]Open in a new tab

   Pyrvinium pamoate impedes the growth of subcutaneous Molm13 and
   Molm13-XR xenograft tumors in mice. (A) The relative tumor volume of
   the Molm13 (left) or Molm13-XR (right) subcutaneous xenografts was
   recorded every 2 or 3 days when DMSO, 0.5 mg/kg pyrvinium, or 0.8 mg/kg
   pyrvinium was administrated by intraperitoneal injection as a function
   of time in the three groups. (B) Comparison of tumor volume of the
   Molm13 (left) and Molm13-XR (right) tumors isolated from each group 13
   and 14 days, respectively, after treatments. (C) Kaplan–Meier survival
   curves of different groups of mice bearing Molm13 (left) and Molm13-XR
   (right) were plotted against days after treatment with pyrvinium or
   DMSO. (D) The body weight of mice bearing Molm13 (left) or Molm13-XR
   (right). Data represent the mean ± SD for each group. * p < 0.05; ** p
   < 0.01. n.s., statistically non-significant.

4. Discussion

   In this study, we revealed that pyrvinium pamoate could not only
   inhibit the FLT3 signaling pathway, but also target mitochondria and
   induce cellular stress responses; these ultimately inhibited cell
   proliferation and triggered the apoptosis of Molm13 and derived Molm13
   (Molm13-XR) myeloid leukemia cells ([138]Supplementary Figure S1). To
   the best of our knowledge, this is the first study to comprehensively
   report the anti-malignant effect of pyrvinium in acute myeloid leukemia
   cells through transcriptomic analysis and extensive functional assays.

   Several anti-cancer mechanisms of pyrvinium have been reported in solid
   tumors, including the regulation of multiple signaling pathways (such
   as β-catenin, AKT, STAT3, and Hedgehog signaling) which support cell
   proliferation and survival, and the regulation of cellular response
   signaling (such as autophagy and UPR), which is observed in several
   solid tumor malignancies [[139]13]; however, reports of the role of
   pyrvinium in myeloid leukemia are limited [[140]12,[141]39]. Harada et
   al. reported that pyrvinium could inhibit ATP production, STAT3
   phosphorylation, and human erythroleukemia (HEL 92.1.7) cell
   proliferation, but did not have this activity in corresponding cells
   without mitochondrial DNA [[142]12]. Another report focused on two
   acute monocytic leukemia cell lines, SHI-1 and THP-1, both of which
   carry the mixed-lineage leukemia (MLL)-rearrangement. They found that
   pyrvinium co-localized with mitochondria in cells and concluded that
   the drug could inhibit mitochondrial respiration and was highly
   effective against MLL-rearranged AML cells in vitro [[143]39]. In this
   study, we evaluated the effect of pyrvinium on mitochondrial functions
   from various aspects and highlighted ISR as an alternative downstream
   message of mitochondrial stress that exerts a cytotoxicity effect
   partly in an STAT5-associated manner.

   Our whole transcriptome analysis further revealed that pyrvinium could
   induce obvious ISR, as demonstrated by the activation of the eIF2α
   -ATF4 pathway, and the subsequent increase in the transcription of the
   downstream genes associated with redox balance (CTH and CHAC1), amino
   acid synthesis and transport (SLC7A11, SLC6A9, ASNS), and metabolic
   reprogramming (GPT2, PCK2, PSAT1). Normal ISR signaling could primarily
   act through a protective mechanism; however, prolonged ISR would
   promote cell death through induction of the expression of a series of
   pro-apoptotic genes, including BBC3 (encodes PUMA) and PMAIP1 (encodes
   NOXA). It is worth noting that after pyrvinium treatment, the
   expression of DDIT3, TRIB3 and CHAC1 genes, which were reported to be
   associated with CHOP-related cell death [[144]36,[145]40,[146]41], was
   stimulated, contributing to pyrvinium-induced apoptosis. In addition,
   ISR was reported to play a crucial role in hematopoietic stem cells and
   contribute to their persistence [[147]42], indicating the importance of
   the optimal regulation of the ISR for the survival of leukemic cells.
   Other series studies investigating ONC201 and ONC212 (imipridone)
   suggested that the upregulation of ISR could promote anti-cancer
   activity against solid and hematological malignancies
   [[148]43,[149]44,[150]45,[151]46], and help overcome the resistance to
   the B-cell lymphoma (BCL)-2 inhibitor venetoclax [[152]47]. Although
   ONC201 and ONC212 exhibit similar effects on the ISR, pyrvinium, being
   an FDA-approved drug, may be more suitable for further clinical trials
   as part of drug repurposing for acute myeloid leukemia.

   The pathway enrichment analysis by Metascape also revealed that
   exposure of cells to pyrvinium caused the downregulation of the ATP
   metabolic process and a decrease in the expression of several genes
   involved in the mitochondria electron transport chain, including
   MT-COX3 (cytochrome c oxidase subunit III), MT-CYB (cytochrome b), and
   MT-ND5 (subunit of complex I). To determine whether pyrvinium-induced
   ISR is associated with mitochondria, we further studied the effect of
   pyrvinium on the mitochondria complex and its function. In this study,
   we demonstrated that pyrvinium co-localized with mitochondria and
   caused mitochondrial stress, presented by the inhibition of complex I
   activity, impediment of mitochondrial OXPHOS, and a decrease in both
   mitochondrial mass and ATP production. Likewise, an antimalarial
   compound atovaquone has also been reported to simultaneously induce ISR
   and suppress OXPHOS [[153]48]. Recently, Hlozkova and others detected
   the basal metabolic profile of several leukemia cell lines, including
   Kasumi-1, Molm13, and MV4-11 cells [[154]49]. Among them, Kasumi-1
   cells had the highest mitochondrial function, with the highest basal
   respiration, ATP-linked respiration, and maximal respiration, while
   Molm13 and MV4-11 cells had similar mitochondrial function. However, in
   our study the IC[50] of pyrvinium pamoate in Kasumi-1 cells was higher
   than that in Molm13 and MV4-11 cells ([155]Figure 1A). In addition, our
   unpublished results showed that the maximal respiration of the
   Molm13-XR cells was significantly higher than that of Molm13 cells, and
   its basal respiration and ATP-linked respiration also tended to be
   higher than that of Molm13 cells. This information indicates that we
   could not conclude that cells with a higher mitochondrial function are
   more susceptible to pyrvinium pamoate. We also evaluated ECAR using the
   glycolysis stress test, which demonstrated increased basal glycolysis
   and glycolytic capacity after treatment with 10 nM pyrvinium in Molm13
   cells. However, the compensation for the upregulated glycolysis rate
   via mitochondria OXPHOS reduction could not rescue the loss of the
   total ATP. In contrast, the compensatory increase in glycolysis was not
   observed in Molm13-XR cells after pyrvinium treatment, which may be due
   to the cell’s original higher glycolytic capacity than that of its
   parental cell line. In addition, we observed that pyrvinium could cause
   the accumulation of ROS in Molm13 cells, and it has been reported to
   selectively trigger the ROS release and mitochondrial membrane
   depolarization, as well as inhibit aerobic glycolysis in KRAS-mutant
   lung cancer cells [[156]50]. Low doses of ROS can play an important
   role in cell cycle progression, proliferation, differentiation, and
   cell migration [[157]51], while higher doses can contribute to ISR
   activation [[158]52]. This information supports our hypothesis
   regarding the connection between mitochondria dysfunction and the
   integrated stress response triggered by pyrvinium.

   GSEA of our transcriptomic profile revealed that a set of genes, which
   are upregulated after amino acid deprivation, were also upregulated
   after pyrvinium treatment, indicating that pyrvinium treatment could
   trigger a cell response similar to that of amino acid deprivation
   ([159]Figure 2C, upper). Further, cancer cells exhibit higher demand
   for amino acids [[160]53] and have better responses to amino
   acid-deprivation therapies targeting amino acid uptake and catabolism
   than normal cells [[161]54]. Furthermore, hematological malignancies
   frequently develop dependency on specific amino acids for their
   survival, and insufficient production of these amino acids may induce a
   metabolic vulnerability and therapeutic opportunities [[162]55]. For
   example, glutaminolysis was identified as synthetically lethal with
   FLT3-TKI treatment, indicating the potency of using amino acid
   depletion in combination therapies [[163]56,[164]57]. Another FLT3-TKI
   gilteritinib was also proved to hinder glutamine uptake and utilization
   in FLT3-ITD–positive AML [[165]58]. Recently, NEI-01, a novel
   arginine-depleting enzyme, could not only induce arginine deprivation,
   but also had cytotoxic activity against arginine auxotrophic AML cells
   through induction of cell cycle arrest and apoptosis [[166]59].
   Moreover, ADI-PEG20, which degrades arginine to citrulline, could
   prevent the proliferation of tumor cells in several cancers [[167]60],
   and a further study showed its efficacy when combined with cytarabine
   in treating AML in mouse models [[168]61]. A phase I study of ADI-PEG20
   plus low-dose cytarabine for the treatment of AML is ongoing [[169]62].
   Given that pyrvinium pamoate induces a similar transcriptomic pattern
   to amino acid deprivation, it is important to determine the amino acids
   whose synthesis is disrupted by pyrvinium pamoate to identify
   alternative metabolic vulnerabilities to develop a combination strategy
   with chemotherapy.

   Furthermore, to explore the mechanism by which pyrvinium induces the
   inhibition of Molm13 cells, several chemicals affecting gene expression
   patterns in a similar manner as pyrvinium were screened using a
   connectivity map [[170]63,[171]64]. Interestingly, androgen receptor
   inhibitors (idazoxan, phenoxybenzamine) [[172]23], K^+ ionophores
   (valinomycin) [[173]65], mTOR inhibitors (sirolimus), and PI3K
   inhibitors (LY-294002) were found to exhibit similar effects on
   genome-wide gene expression as those of pyrvinium ([174]Supplementary
   Figure S2; [175]Table S4).

   We demonstrated the downregulation of the protein folding pathway,
   which includes several HSP genes, such as HSP90B1, HSPA, HSPA8, HSPH1,
   and DNAJA1 (HSPF4). Activation of HSF1, which transcriptionally
   triggers the mRNA expression of HSPs [[176]66], was significantly
   inhibited by pyrvinium, as demonstrated by GSEA ([177]Figure 2C,
   lower). Previous research suggested that HSF1 activation would be
   inhibited by amino acid starvation [[178]67] or mTOR inhibition
   [[179]68]. Finally, we discovered that pyrvinium could induce apoptosis
   in both Molm13 and Molm13-XR cells (a cabozantinib-resistant Molm13
   cell line), indicating that targeting mitochondrial respiration and
   ISRs might not only be effective in the treatment of AML, but also in
   overcoming cabozantinib resistance. Further transcriptome comparison
   indicated a little overlap between cabozantinib-resistant DEGs and
   pyrvinium-induced DEGs ([180]Supplementary Figure S3), which may be the
   reason why Molm13-XR cells were still sensitive to pyrvinium.

5. Conclusions

   In summary, this study provides an example of a drug repurposing
   strategy. We found that pyrvinium could localize to mitochondria, block
   complex I activity, elevate ROS levels, and then trigger a
   pro-apoptotic ISR in both FLT3-ITD-positive Molm13 cells and
   cabozantinib-resistant Molm13-XR cells. These findings suggest that
   pyrvinium can be used in the treatment of AML in the future.

Acknowledgments