Anti-leukemia effects of Omipalisib in Acute Myeloid Leukemia: inhibition of PI3K-AKT-mTOR signaling and suppression of Mitochondrial Biogenesis

Omipalisib (GSK2126458), a potent dual PI3K/mTOR inhibitor, is reported to exhibit anti-tumor effect in several kinds of cancers. More than 50% of acute myeloid leukemia (AML) patients display a hyperactivation of PI3K/AKT/mTOR signaling. We investigated the anti-proliferative effect of omipalisib in AML cell lines with varied genetic backgrounds. The OCI-AML3 and THP-1 cell lines had a signi�cant response to omipalisib, with IC 50 values of 17.45 nM and 8.93 nM, respectively. We integrated transcriptomic pro�le and metabolomic analyses, and followed by gene set enrichment analysis (GSEA) and metabolite enrichment analysis. Our �ndings showed that in addition to inhibiting PI3K/AKT/mTOR signaling and inducing cell cycle arrest at the G 0 /G 1 phase, omipalisib also suppressed mitochondrial respiration and biogenesis. Furthermore, omipalisib downregulated several genes associated with serine, glycine, threonine, and glutathione metabolism, and decreased their protein and glutathione levels. In vivo experiments revealed that omipalisib signi�cantly inhibited tumor growth and prolonged mouse survival without weight loss. Gedatolisib and dactolisib, another two PI3K/mTOR inhibitors, exerted similar effects without affecting mitochondria biogenesis. These results highlight the multifaceted anti-leukemic effect of omipalisib, revealing its potential as a novel therapeutic agent in AML treatment.


Introduction
Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by clonal expansion and impaired cell differentiation.Chromosomal alterations and mutations in leukemic cells are critical for their dysregulated survival and proliferation [1,2].Recently, owing to the understanding of the molecular landscape of AML through large-scale genomic analysis, the development of targeted therapies has greatly improved [3].Although some new drugs, including FLT3 inhibitors (midostaurin and gilteritinib), IDH1/2 inhibitors (enasidenib and ivosidenib), and BCL2 inhibitor (venetoclax), have been approved by the FDA for the treatment of speci c subgroups of AML [4], the development of effective and durable therapeutic strategies for other subgroups of AML remains unmet.

Cell viability assay and ow cytometry
Following treatment with drugs for 72 h, cell viability was assessed by the Colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) as previously described [27].Each experiment was performed in triplicates.
For cell cycle analysis, following treatment with the drug for 24 h, the cells were xed in 70% ethanol, and stained with propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA).To detect cell apoptosis, a Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Biosciences) was used.Following treatment with the drug for 72 h, the cells were stained with Annexin V-FITC and PI.All uorescence intensities were measured using a CYTOFLEX TM Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA).Cell cycle distribution and apototic cells were analyzed using FlowJo 10.4 software

Western blot analysis
Following treatment with the drug for the indicated times, total cell lysates were separated on SDS-PAGEand then transferred to PVDF membranes (Millipore, Billerica, MO, USA).After blocking with BlockPRO™ 1 Min Protein-Free Blocking Buffer (Energenesis Biomedical Co., Taiwan), membranes were incubated with indicated primary antibodies (Table S1) in 4°C for overnight.Membranes were then washed with TBS and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) for 60 min at RT. Immunoblot signals were detected by chemiluminescence using Western Lightening Plus-ECL (Perkin Elmer, Waltham, MA, USA) and ChemiDoc™ XRS (Bio-Rad Laboratories).
RNA extraction, RNA-sequencing (RNA-seq), and quantitative RT-PCR Total RNA was extracted using NucleoSpin® RNA extraction kit (Macherey-Nagel, Düren, Germany).RNAseq was performed using an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA) and subsequent bioinformatics analysis was performed as previously described [27].Brie y, a total amount of 1μg RNA was used for library preparation.Library quality was assessed using an Agilent Bioanalyzer 2100 system with DNA High-Sensitivity Chips.Sequencing alignment of hg19 was performed using Rsubread (2.1.0),and differentially expressed gene (DEG) identi cation was conducted with EBSeq (1.26.0).These transcriptome les were posited in the Gene Expression Omnibus (GEO) database (ID: GSE224155).Pathway enrichment was then analyzed by gene set enrichment analysis (GSEA) (4.0.3) using the Molecular Signatures Database v7.0 (MSigDB, Broad Institute, Cambridge, MA, USA) and Metascape.cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scienti c).
The qRT-PCR was performed and analyzed on a QuantStudio 3 Real-Time PCR System (ThermoFisher Scienti c) with 2x qPCRBIO SyGreen Blue Mix Lo-ROX (PCR Biosystems, London, UK).The primers used for speci c gene detection are listed in Table S2.

Mitochondria analysis and mtDNA quantification
Mitochondrial mass, ROS content, and mitochondrial membrane potential in cells were measured by CYTOFLEXTM Flow Cytometer (Beckman Coulter) using MitoTracker Green, MitoSOX kit, and TMRE (Invitrogen/ThermoFisher Scienti c, Waltham, MA, USA), respectively, according to the manufacturer's instructions.
To determine the mtDNA copy number, total DNA was isolated from cell lines or tissues using DNeasy kits (Qiagen, Venlo, Netherlands).Nuclear and mitochondrial DNA content was analyzed using qPCR.The primers used for qPCR are listed in Table S2.
A Jet Stream electrospray ionization source with a capillary voltage of 4.0 kV was used in positive mode for sample ionization.The MS parameters were set as follows: gas temperature, 325 °C; gas flow, 5 L/min; nebulizer pressure, 40 psi; sheath gas temperature, 325 °C; and sheath gas flow, 10 L/min.The scan range was set at 50-1700 m/z.Metabolite intensities were analyzed using z-transformation.Analysis of the targeted metabolomics dataset was performed by using MetaboAnalyst 5.0.

Cellular bioenergetic analysis
Oxygen consumption rate (OCR) measurements were performed using a Seahorse an XFe24 extracellular ux Analyzer (Agilent Technologies/Seahorse Bioscience, Billerica, MA, USA) according to the manufacturer's instructions.Cells were pretreated with drug for 24 h and seeded in a 24-well cell culture microplate precoated with Cell-Tak® Cell and Tissue Adhesive (Corning, Corning, NY).The Cell Mito Stress Test Kit (Agilent Technologies/Seahorse Bioscience) was used to measure mitochondrial respiration.After baseline measurements, oligomycin A, FCCP, rotenone, and antimycin A were sequentially applied to the microplate.The results were analyzed using the wave 2.4 (Agilent Technologies/Seahorse Bioscience).
In vivo e cacy of omipalisib in a murine model A proper amount of OCI-AML3 cells were subcutaneously inoculated into CAnN.Cg-Foxn1 nu /CrlNarl mice (National Laboratory of Animal Breeding and Research Center, Taipei, Taiwan).When the tumor size reached 100 mm 3 , the mice were randomly divided into three groups, one receiving oral omipalisib with 0.2 mg/kg (n = 10), the other one receiving oral omipalisib of 1 mg/kg (n = 11) and one vehicle group (n = 11).Treatments were administered for two days, followed by a 1-day rest, for a total of seven cycles.
All the mice were regularly monitored for body weight and tumor growth.Tumor volumes were calculated using the following formula: (length × width 2 ) × 0.5 in millimeters.The mice were euthanized for further analysis when the tumor volume reached 1500 mm

Results
Omipalisib potently suppressed AML cell proliferation We investigated the activity of the PI3K/AKT/mTOR signaling pathway in various leukemia cell lines.
Transcriptomic analysis revealed that omipalisib downregulated amino acid metabolisms and mitochondrial biogenesis in AML We performed RNA sequencing (RNA-seq) to compare omipalisib-and DMSO-treated OCI-AML3 cells after exposure to the drug for 24 h.We identi ed a total of 2056 differentially expressed genes (DEGs), of which 811 were downregulated and 1245 were upregulated (p < 0.05 and -1 ≥ log 2 FC ≥ 1) post omipalisib treatment for 24 h (Fig. 2A).Interestingly, the most suppressed genes after omipalisib treatment were those associated with the cell cycle, DNA metabolic processes, PI3K/AKT/mTOR signaling, the MYC pathway, and E2F pathway (Fig. 2B).Gene set enrichment analysis (GSEA) further showed marked suppression of the KEGG pathway gene sets, including "Gly, Ser and Thr metabolism", "Cys and Met metabolism", "Carbon metabolism", and "Oxidative phosphorylation" following omipalisib treatment (Fig. 2C).Furthermore, we found that gene sets that contain genes encoding components of mitochondrial biogenesis and the respiratory electron transporter chain were signi cantly suppressed in the omipalisib-treated group (Fig. 2D).Based on the results of these transcriptomic analyses, we proposed that omipalisib not only suppresses cell proliferation through PI3K/AKT/mTOR signaling inhibition but also reprograms cellular metabolisms and impairs mitochondrial biogenesis.

Omipalisib reduced oxidative phosphorylation and impaired mitochondrial biogenesis in AML
To examine the effects of omipalisib on mitochondrial biogenesis and oxidative phosphorylation, a Seahorse XF Cell Mito Stress Assay was performed.We found a signi cant decrease in basal OCR, maximal respiration, spare-respiratory capacity, and proton leak-driven OCR in omipalisib-treated OCI-AML3 cells (Fig. 3A).Similar results were observed in THP-1 cells (Fig. S2A).Flow cytometry analysis revealed that omipalisib decreased mitochondrial membrane potential and mitochondrial mass but increase mitochondrial ROS in OCI-AML3 cells (Fig. 3B).In addition, qRT-PCR results demonstrated that omipalisib signi cantly decreased the mtDNA content (Fig. 3C) and inhibited the expression of genes associated with mitochondrial biosynthesis, including PPARGC1B, TFAM, and NUDFS6 (Fig. 3D).Immunoblot analysis indicated that the protein levels of PGC1β were signi cantly decreased in OCI-AML3 cells after 72 h omipalisib treatment (Fig. 3E).These results indicated that omipalisib suppressed mitochondrial respiration and inhibited mitochondrial biogenesis in myeloid leukemia cells.

AML cells exhibit metabolic changes in response to omipalisib
To determine the role of metabolic reprogramming in response to omipalisib treatment, we performed a metabolomic analysis of OCI-AML3 cells after omipalisib treatment for 24 h.In total, 82 metabolites were identi ed in the targeted metabolomic analysis.PCA demonstrated a clear separation between the omipalisib-and the DMSO-treated group (Fig. 4A).Of the 82 metabolites, 22 signi cantly increased or decreased (p-value ≤ 0.05) after omipalisib treatment compared to the DMSO controls (Fig. 4B).
Metabolomics analyses indicated that omipalisib treatment signi cantly decreased the intracellular levels of L-valine, L-alanine, L-tryptophan, L-phenylalanine, L-tyrosine, L-histidine, and pyruvate (Fig. 4C and Table S3).Metabolite set enrichment analysis (MSEA) revealed that these metabolites are involved in the biosynthesis and degradation of various amino acids (Fig. 4D and Table S4).These results indicate that omipalisib suppressed amino acid metabolism in OCI-AML3 cells.
Omipalisib inhibited glutathione metabolism through suppressing de novo serine synthesis and folate/methionine cycle in AML Joint-Pathway Analysis (MetaboAnalyst 5.0) which combined transcriptomic and metabolomic data revealed that "glutathione metabolism" (p-value = 0.000278, impact = 0.36548) and "glycine, serine, and threonine metabolism" (p-value = 0.004847, impact = 0.25803) were the most signi cantly downregulated pathways in the omipalisib-treated group (Fig. 5A and Table S5).Subsequently, qRT-PCR analysis con rmed that the expression of several genes related to de novo serine synthesis pathway, regulators of glycine biosynthesis, glutathione synthesis, and glycolysis was signi cantly decreased in omipalisibtreated OCI-AML3 cells (Fig. 5B).Similar results were obtained in THP-1 cells (Fig. S2B).In concordance with the transcriptomic alternations, the metabolomic pro ling data also showed that the cellular levels of glutathione and pyruvate were signi cantly decreased (Fig. 5C).Immunoblotting analysis indicated that the expression of PGHDH and PSAT1 was signi cantly decreased in OCI-AML3 cells after 24 h of omipalisib treatment (Fig. 5D).In summary, we propose that omipalisib inhibits glycolysis, de novo serine synthesis, the folate/methionine cycle, and glutathione, resulting in decreased glutathione and pyruvate levels (Fig. 5E).
Gedatolisib and dactolisib exerted similar effects on glutathione metabolism and oxidative phosphorylation in AML Dactolisib (BEZ-235) and gedatolisib (PF-05212384), two other dual PI3K/mTOR inhibitors, have been reported to suppress proliferation and migration and reverse multidrug resistance in AML [17,18].These two inhibitors suppressed the expression of several genes associated with de novo serine synthesis, the folate/methionine cycle, glutathione synthesis, and glycolysis (Fig. S3A, B).Gedatolisib suppressed basal OCR, proton leak, and maximal respiration in OCI-AML3 cells (Fig. S3C).Moreover, gedatolisib increased mitochondrial ROS and decreased mitochondrial membrane potential but it did not suppress mitochondrial mass and mtDNA content in OCI-AML3 cells (Fig. S3D, E).In addition, gedatolisib and dactolisib did not suppress the expression of mitochondrial biosynthesis-related genes (Fig. S3F).These results indicated that gedatolisib and dactolisib could only suppress glutathione metabolism and mitochondrial respiration rather than mitochondrial biogenesis.

Omipalisib suppressed the growth of subcutaneous OCI-AML3 xenograft tumors
To evaluate the anti-leukemic e cacy of omipalisib in vivo, OCI-AML3 xenografted nude mice were treated with vehicle (DMSO) or omipalisib (0.2 mg/kg or 1 mg/kg).Omipalisib was well-tolerated by the mice, and there were no side effects on body weight during the dosing period (Fig. 6A).In line with the in vitro results, mice treated with 0.2 or 1 mg/kg omipalisib showed retarded OCI-AML3 tumor growth (Fig. 6B, C).Compared with the vehicle group, oral administration of 0.2 or 1 mg/kg omipalisib signi cantly prolonged the survival of mice bearing OCI-AML3 tumors (Fig. 6D).

Discussion
This study demonstrates the potent anti-leukemic effect of omipalisib in vitro and in vivo.While its effects on other cancers [22][23][24][25][26] have been studied, its impact on leukemia was previously unknown.Our results show that omipalisib effectively inhibits PI3K/AKT/mTOR signaling pathway, triggers cell cycle arrest, and regulates metabolic pathways in AML cells.This is the rst study to investigate the antimalignant activity of omipalisib and its detailed mechanisms in AML by integrating transcriptome, metabolome, and functional assays.
Our analysis of metabolomic and transcriptomic data revealed that omipalisib suppressed de novo serine synthesis, the folate/methionine cycle, and glutathione metabolism, which are dysregulated in various cancers [28-31], including FLT3-ITD-driven AML [32].These metabolic pathways are critical for protein, nucleotide and lipid synthesis, generation of glutathione and NADPH, and methylation reactions [33,34].
Omipalisib and other PI3K/mTOR dual inhibitors signi cantly suppressed the expression of genes involved in these metabolic pathways, including PHGDH, PSAT1, PSPH, SHMT1/2, and MTHFD1/2.Dysregulation of serine metabolism through the mTORC1-ATF4 axis has also been reported in FLT3-ITD AML [32], highlighting the potential of serine metabolism as treatment target in AML.
Notably, we found that omipalisib, but not gedatolisib or dactolisib, suppressed oxidative phosphorylation and disrupted mitochondrial biogenesis.We demonstrated that omipalisib signi cantly inhibited mitochondrial respiration rate, decreased membrane potential, and increased mitochondrial ROS.
Furthermore, omipalisib strongly decreased mitochondrial mass, mtDNA, and the expression of several genes related to mitochondrial biogenesis, as well as their protein levels.Additionally, AML cells have higher mitochondrial biogenesis than normal hematopoietic cells [35,36], and AML progression requires increased mitochondrial biogenesis and oxidative phosphorylation [37,38].Therefore, mitochondrial metabolism and biogenesis are potential therapeutic targets for AML treatment.
In this study, we elucidated the cytotoxicity and metabolic alterations caused by omipalisib in AML, both in vitro and in vivo.The combination of metabolomic pro ling and transcriptomic analyses, followed by various precise experiments, uncovered the molecular mechanisms of omipalisib, including the inhibition of serine and glutathione biosynthesis and impairment of oxidative phosphorylation and mitochondrial biogenesis in AML.Recently, a randomized, placebo-controlled study demonstrated acceptable tolerability of omipalisib in idiopathic pulmonary brosis [53].Participants receiving 2.0 mg omipalisib twice daily achieved a maximum observed concentration (C max ) of 170 nM, which is well above the effective dose used in this study.To date, omipalisib has been demonstrated to be an effective therapeutic strategy in combination with CDK4/6 and autophagy inhibitors for the treatment of various cancer types [23,24].Omipalisib has the advantages of sensitizing cancer cells to radiotherapy and chemotherapy [22,54].Therefore, combined treatment strategy using omipalisib is an effective future protocol for AML.

Declarations Figures
In Omipalisib suppressed serine biosynthesis and impaired glutathione metabolism A Joint Pathway Analysis matched according to p-values from pathway enrichment analysis (y-axis) and pathway impact values from pathway topology analysis (x-axis).The color and size of each circle are based on p-values and pathway impact values, respectively.Small pvalues and large pathway impact circles indicate that the pathway was signi cantly perturbed.B Expression of genes associated with de novo serine synthesis, folate/methionine cycle, glutathione metabolism, and glycolysis-related genes in OCI-AML3 cells treated with 50 nM omipalisib for 24 h.
vitro effects of omipalisib and gedatolisib on OCI-AML3 and THP-1 myeloid leukemia vitro effect and IC 50 of Omipalisib and gedatolisib on various myeloid leukemia cells.Data are representative of two independent experiments each performed in triplicate.C-D Immunoblot analysis of the indicated proteins in OCI-AML3 cells and THP-1 cells treated with DMSO or 10 nM, or 50 nM omipalisib for 4 h and probed with antibodies to pAKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, p-4EBP1, and 4EBP1.β-actin was used a loading control.Representative immunoblots of three independent experiments are shown.E-F After exposure to indicated concentrations of omipalisib for 24 h, various populations of OCI-AML3 cells and THP-1 cells were analyzed by ow cytometry using propidium iodide (PI) staining.***p < 0.001, **p < 0.01, *p < 0.05 vs. DMSO control.G-H Immunoblot analysis of the indicated proteins in OCI-AML3 and THP-1 cells treated with DMSO, 10 nM omipalisib, or 50 nM omipalisib for 4 h and probed with anti-p27 antibodies.β-actin was used as a loading control.

Figure 4 Metabolomic
Figure 4 Transcript levels were normalized to those of 18s rRNA.The relative mRNA expression was calculated using the 2 −ΔΔCT method.Each column represented the averages ± SD of three independent experiments.***p < 0.001, **p < 0.01, *p < 0.05, compared with DMSO control.C Levels of glutathione (left) and pyruvate (right) in omipalisib-treated OCI-AML3 cells as measured by untargeted metabolomics.***p < 0.001, *p < 0.05, compared with DMSO control.D Immunoblot analysis of proteins associated with de novo serine synthesis in OCI-AML3 cells exposed to various concentrations of omipalisib for 4 h and probed with anti-PHGDH and anti-PSAT1 antibodies.GAPDH was used as a loading control.Representative immunoblots of three independent experiments are shown.Values represent the fold change with respect to the DMSO control group, after normalization to α-tubulin.E Schematic representation of metabolic pathways including glycolysis, the TCA cycle, de novo serine synthesis, the folate/methionine cycle, and glutathione metabolism.
3. The xenograft experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, National Taiwan University (#20180457, 30 Jan 2019), and conformed to 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.
Statistical analysisStatistically signi cant differences were evaluated using a two-sided Student's t-test.Error bars indicate means ± S.D. or means ± S.E.M. of at least three independent triplicate experiments.The IC 50 values were determined using the AAT Bioquest IC 50 calculator Principal component analysis (PCA) was performed by using ClustVis.P values of less than 0.05 were considered statistically signi cant.