Reagents

Standard reagents were obtained from Themo Fisher Scientific (MA, USA) and Sigma-Aldrich (MO, USA). KL-11743 and KL-12023 were kindly provided by Kadmon Pharmaceuticals (NY, USA). Oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, antimycin A, aminooxyacetic acid, etomoxir, cycloheximide, phenylsuccinic acid, 2-(aminooxy)acetic acid, 3-nitropropionic acid, and thenoyltrifluoroacetone were from Sigma-Aldrich. ABT-737, IACS-010579, and GSK-1120212 were from Selleck Chemicals (TX, USA). MitoTracker™ Green FM, Tetramethylrhodamine ethyl ester, and DAPI were from Thermo Fisher Scientific. All primary cell culture additives were from Stem Cell Technologies (MA, USA).

Cell models

NB4, THP-1, and MOLT-4, lines were obtained from American Type Cell Culture. Cells were cultured in Roswell Park Memorial Institute Medium (RPMI-1640) containing phenol red and 300 mg/L L-glutamine (Thermo Fisher Scientific) and supplemented with 10% heat inactivated fetal bovine serum, 100 U/mL penicillin, and 100 ug/mL streptomycin (Thermo Fisher Scientific). Glutamine deficient RPMI media was purchased from the Memorial Sloan-Kettering Cancer Center Media Preparation Facility. Cells were incubated in a humidified incubator at 37 °C with 5% CO2 and confirmed to be mycoplasma-free by the HEK-Blue Detection Kit (Invitrogen, MA, USA). Induced pluripotent stem cell (iPSC) lines encompassing normal cells (N-8.2), KRASWT AML (AML-4.24), and KRASG12D (AML-4.10) AML were generated, cultured and differentiated into HSPCs/LSCs as previously described [27, 28]. Patient AML samples were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 1% penicillin, 1% streptomycin, 2 mM glutamine, 5 μM β-mercaptoethanol, 20 ng/ml hIL-3, 50 ng/mL hIL-6, 20 ng/ml hGM-CSF, 20 ng/ml hG-CSF, 20 ng/ml hTPO, and 25 ng/mL hSCF. De-identified patient samples were provided by the Tisch Cancer Institute’s Hematological Malignancies Tissue Bank through an Institutional Review Board at the Mount Sinai School of Medicine approved protocol (STUDY-11–02054-MOD008; PI: Bridget Marcellino).

Glucose uptake assays

Cells were seeded in 96-well plates in quadruplicate and treated with DMSO or KL-11743 (500 nM). After 24 h, 200 μL of cell suspension was removed and centrifuged at 400 × g for 5 min. Resulting cell pellets were resuspended in fresh media, transferred back to their respective wells, and normalized by YOYO3 staining, described below. Supernatant was used to measure glucose content using the Glucose (GO) assay kit (Sigma-Aldrich) scaled down to be assessed on a 96-well tissue culture plate and performed according to the manufacturer’s instructions. Absorbance readings were measured at 540 nm using a plate reader (Synergy H1 Hybrid multi-mode micro-plate reader, Biotek/Agilent, CA, USA). Concentrations were determined by a standard curve, then normalized to cell count and calculated as a percentage relative to base glucose levels in culture media containing 10% FBS.

Nutrient consumption analysis

Cells were treated with KL-11743 (500 nM) or DMSO. At 24 h, glucose, lactate, glutamine, and glutamate concentrations in the medium were measured with an YSI7000 electrochemical analyzer (YSI) in collaboration with the Donald B. and Catherine C. Marron Cancer Metabolism Center at Memorial Sloan Kettering Cancer Center (NY, USA). Absolute values for consumption/secretion of metabolites were calculated by subtracting the concentration in medium incubated for 24 h without cells, and rates were derived by normalizing to cell number, media volume, and hours of incubation [29,30,31].

Amino acid consumption analysis

Cells were treated with KL-11743 (500 nM) or DMSO for 24 h and amino acid concentrations were determined by GC–MS-EI in collaboration with the Metabolomics Core at the Icahn School of Medicine at Mount Sinai. For metabolite measurements from culture medium, 50 μL of cell-conditioned medium was extracted by the addition of 200 μL ice-cold methanol and stored at -80 °C overnight. 50 μL of no-cells medium incubated for the same amount of experimental time was processed in parallel and used as a reference to determine metabolite secretion or consumption. The methanol-extracted metabolites were cleared by centrifugation at 20,000 × g for 20 min 4 °C, and supernatants was dried in a vacuum evaporator (Genevac EZ-2 Elite) for 2 h. Dried metabolites were dissolved in 20 mg/mL of methoxyamine hydrochloride (Sigma-Aldrich) in pyridine (Thermo Fisher Scientific) for 90 min at 30 °C and derivatized with MSTFA with 1% TMCS (Thermo Fisher Scientific) for 30 min at 37 °C. Samples were analyzed using an Agilent 7890A GC connected to an Agilent 5975C Mass Selective Detector with electron impact ionization.

Normalization for cell count

Cells were treated with 0.1% Triton and 0.125 μM YOYO™-3 Iodide (Invitrogen, MA, USA) and incubated at room temperature overnight, protected from light. 599/640 nm (excitation/emission) wavelength was measured with Synergy H1 Hybrid multimode microplate reader (BioTek/Agilent). Fluorescence unit values were divided by the 20% trimmed mean calculated from all wells to obtain normalization factors.

Cell counts for proliferation & G1-phase cell cycle arrest analysis

Cells were plated at an initial concentration of 2 × 105 cells/mL and treated with 500 nM KL-11743 or an equal volume of DMSO. Every 12 h, a portion of the cell suspension was removed, pelleted, resuspended in a smaller volume, and counted using a hemocytometer. Trypan Blue Solution (0.4%) (Thermo Fisher Scientific) was used at a 1:1 ratio to stain and exclude dead cells. For G1-phase cell cycle arrest quantification, cells were treated as indicated, trypsinized, washed with PBS, and resuspended in Nicoletti-Buffer (0.1% TX-100, 0.1% sodium citrate, 50 μg/ml propidium iodide). Intact nuclei were analyzed by flow cytometry to quantify G1 DNA content. Data analysis was conducted using FCS Express 7 computer software. Unless indicated otherwise, flow cytometry data was collected by the Flow Cytometry CoRE at the Icahn School of Medicine at Mount Sinai.

EdU flow cytometry assays

Percentages of cells proliferating were obtained using the Click-iT™ EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Invitrogen) and prepared according to manufacturer’s instructions. Briefly, cells were treated as indicated for 24 h, followed by a 2 h incubation with 10 μM EdU at 37 °C. Cells were harvested, washed, permeabilized, and fixed, and analyzed by flow cytometry at 633/635 nm excitation. Data analysis was conducted using FCS Express 7 computer software. Unless indicated otherwise, flow cytometry data was collected by the Flow Cytometry CoRE at the Icahn School of Medicine at Mount Sinai.

Apoptosis assays

Cell lines were treated in 12-well plates as indicated, with at least 3 × 105 cells per sample. The day of the assay, cell suspensions were transferred to 12 × 75 mm polystyrene flow cytometry tubes, pelleted at 1000 × g for 10 min at 4 °C and resuspended in 300 μL Annexin-V Binding Buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing ~ 40 ng/μl AlexaFluor 488-conjugated Annexin V. Samples were analyzed for percent positive events by flow cytometry. Data analysis was conducted using FCS Express 7 software. Unless indicated otherwise, flow cytometry data was collected by the Flow Cytometry CoRE at the Icahn School of Medicine at Mount Sinai. Patient AML samples were treated as indicated and cell death was measured using an MTT assay (Sigma-Aldrich) according to the manufacturer’s instructions. This minimized harvesting/labelling stress and processing times to provide more accurate datasets compared to Annexin V analysis of primary cells. Sample were analyzed by measuring the A590 and subtracting the A690 as a reference; % viability was calculated by normalizing absorbance values to DMSO (the negative control) and cycloheximide (the positive control) to 100 and 0%, respectively.

Cell line viability assays

Cells were grown in complete RPMI, glutamine deficient RPMI, serine deficient RPMI, or double deficient RPMI media with DMSO or KL-11743 as indicated in a 96-well plate at 3 × 104 cells per well. CellTiter-Glo 2.0 (Promega) was used to measure the viability after 24 h following the manufacturer’s protocol. Measurements were obtained using an Agilent BioTek Synergy H1 plate reader. Normalization was performed using cycloheximide (50 μg/mL) and ABT-737 (1 μM) co-treatment as 0% viable due to consistent 100% cell death, and DMSO as 100% viable.

Single-cell and Population-level Analyses using Real-time Kinetic Labeling (SPARKL)

Cells were seeded onto poly-D-lysine (Thermo Fisher Scientific)-coated 96-well plates at 3 × 104 cells per well. Cells were treated as indicated in addition to 1 μM cell viability dye YOYO™-3 Iodide (Invitrogen) before immediately subjecting the plate to real-time cell-death analysis. Cells were incubated in a humidified and gas-controlled environment and imaged using a tandem BioSpa and Cytation 7 Cell Imaging Multimode Reader (BioTek/Agilent) as described previously [32]. A 1043 × 1043 μm image was taken per well, analyzed for fluorescently positive objects, and data is reported as the number of positive objects detected per image. Images were collected using a 10 × objective, a laser auto focus module (cat. no. 1225010), and the "Texas Red" filter cube – excitation: 586/15, emission: 647/57 (cat. no. 1225102, BioTek/Agilent).

NAD+/NADH analyses

NAD+ and NADH concentrations were determined using the EnzyChrom™ NAD+/NADH Assay Kit (BioAssay Systems, CA, USA). 1 × 106 cells were treated at a concentration of 2 × 105 cells/mL. Cells were pelleted, resuspended in NAD+ or NADH extraction buffer, and analyzed following manufacturer’s instructions. Optical density was read for time “zero” at 520–600 nm and again after a 15 min incubation at room temperature. Measurements from time “zero” were subtracted from the final reads, and NAD+/NADH concentrations were determined by plotting measurements against a standard curve of known NAD concentrations.

Targeted metabolomics

NB4 (Fig. S2): NB4 was treated with DMSO or KL-011743 (500 nM, 24 h), pelleted, and frozen at -80 °C. Samples was thawed on ice, 100 μL of ultrapure water was added to resuspend the cell pellet. Divide 50 μL cell suspension and add 200 μL of methanol (precooled at -20 °C) and vortexed for 2 min under the condition of 2500 r/min. The sample was frozen in liquid nitrogen for 5 min, removed on ice for 5 min, after that, the sample was vortexed for 2 min. The previous step was repeated for 3 times. The sample was centrifuged at 12,000 r/min for 10 min at 4 °C. Take 200 μL of supernatant into a new centrifuge tube and place the supernatant in -20 °C refrigerator for 30 min. Then the supernatant was centrifuged at 12,000 rpm for 10 min at 4 °C. The sample extracts were analyzed using an LC–ESI–MS/MS system (Waters ACQUITY H-Class, https://www.waters.com/nextgen/us/en.html; MS, QTRAP® 6500 + System, https://sciex.com) by Metware Biotechnology. Metabolites were quantified by multiple reaction monitoring (MRM) using triple quadrupole mass spectrometry. In MRM mode, the first quadrupole screened the precursor ions for the target substance and excluded ions of other molecular weights. After ionization induced by the impact chamber, the precursor ions were fragmented, and a characteristic fragment ion was selected through the third quadrupole to exclude the interference of non-target ions. After obtaining the metabolite spectrum data from different samples, the peak area was calculated on the mass spectrum peaks of all substances and analyzed by standard curves. Profiling Of Widely Targeted Small Metabolites By QTRAP 6500 + LC–MS/MS (Figs. 3, S3A): NB4 and MOLT4 were treated with DMSO or KL-011743 (500 nM, 24 h), pelleted, washed with 1 mL of 150 mM ammonium acetate, pelleted again, and flash-frozen in liquid nitrogen. Samples were screened for alterations in metabolite levels of the Widely Targeted Small Polar Metabolite (WTSM) panel by the Stable Isotope and Metabolomics Core Facility at Albert Einstein College of Medicine, as previously described [33].

KEGG pathway enrichment analysis

Rich Factor for each pathway, the ratio of the number of differential metabolites in the corresponding pathway to the total number of metabolites annotated in the same pathway, was calculated. The greater the Rich Factor, the greater the degree of enrichment. P-value is the calculated using hypergeometric test as shown below:

$$P=1-\sum_^\frac\right)\left(\genfrac\right)}\right)}$$

N represents the total number metabolites with KEGG annotation, n represents the number of differential metabolites in N, M represents the number of metabolites in a KEGG pathway in N, and m represents the number of differential metabolites in a KEGG pathway in M.

Real-time reverse transcription polymerase chain reaction

Cells were treated as indicated for 24 h, then harvested and pelleted by centrifugation for 5 min at 400 × g. Total RNA was extracted from cell pellets with RNeasy Mini Kit (Qiagen, MD, USA), following manufacturer’s instructions. RNA was quantified using a NanoDrop One Spectrophotometer, and 2 μg was used to synthesize cDNA using the RNA to cDNA EcoDry™ PreMix (Double Primed) (Takara Bio, CA, USA). Reaction tubes were placed in a thermocycler and subjected to 42 °C for 1 h and 70 °C for 10 min, followed by an indefinite 4 °C hold until moved to storage at -20 °C. Forward and reverse primers for genes of interest (Table 1) were combined with Power SYBR™ Green PCR Master Mix (Applied Biosystems, CA, USA), and gene expression was analyzed using a ViiA 7 Real-Time PCR system. The expression of relevant genes was normalized to 18S (Table 1).

Table 1 qPCR primer sequences for probed genesAgilent bioanalyzer glycolysis stress test analysis

Cells were treated as indicated at a concentration of 2 × 105 cells/mL. One day prior to running the assay, Agilent XFe96 Sensor Cartridges were hydrated with XF Calibrant, pH 7.4 (Agilent). The day of the assay, cells were pelleted and resuspended in XF RPMI (Agilent) supplemented with 2 mM L-glutamine and the indicated treatment condition. Cells were counted and seeded (1 × 105 cells/well) onto poly-D-lysine (Thermo Fisher Scientific)-coated plates (Agilent) and centrifuged at 200 × g for 3 min. Plates were incubated in a non-CO2 incubator at 37 °C for 40–60 min. OCR and ECAR were measured using the Agilent XFe96 Extracellular Flux Analyzer and the XF Glycolysis Stress Test Kit (Agilent) according to the manufacturer’s instructions. ECAR measurements were determined before and after administration of glucose (10 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (50 mM). At the end of the assay, normalization values for cell count were obtained as described above (“Normalization for Cell Count”), and OCR and ECAR measurements were normalized against these factors.

Agilent bioanalyzer XF cell mito stress test

Cells were treated as indicated at a concentration of 2 × 105 cells/mL. One day prior to running the assay, Agilent XFe96 Sensor Cartridges were hydrated with XF Calibrant, pH 7.4 (Agilent). The day of the assay, cells were pelleted and resuspended in XF RPMI (Agilent) supplemented with 1 mM pyruvate (Agilent), 10 mM glucose (Agilent), 2 mM L-glutamine, as well as the treatment condition. Cells were counted and seeded (1 × 105 cells/well) onto poly-D-lysine-coated plates (Agilent) and centrifuged at 200 × g for 3 min. Plates were incubated in a non-CO2 incubator at 37 °C for 40–60 min. OCR and ECAR were measured using the Agilent XFe96 Extracellular Flux Analyzer and the Agilent XF Cell Mito Stress Test (Agilent) according to the manufacturer’s instructions. OCR was measured 3 times before and after administration of each of the following: oligomycin (1 μM), FCCP (1 μM), and a combination of rotenone and antimycin A (0.5 μM). At the end of the assay, normalization values for cell count were obtained as described above, and OCR and ECAR measurements were normalized against these values.

Agilent bioanalyzer complex I/II activities

Cells were treated as indicated at a concentration of 2 × 105 cells/mL. One day prior to running the assay, Agilent XFe96 Sensor Cartridges were hydrated with XF Calibrant, pH 7.4. Cells were pelleted and resuspended in mitochondrial assay buffer (MAS: 220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% fatty acid-free BSA). Cells were counted and seeded (1 × 105 cells/well) onto poly-D-lysine-coated plates (Agilent), centrifuged at 200 × g for 3 min, and incubated at 37 °C for 30 min. 100 μL MAS was removed from each well and replaced with 100 μL MAS containing plasma membrane permeabilizer for a final concentration of 1 nM in the well. Sensor Cartridge was loaded with 10 × stocks of substrates and/or inhibitors diluted in MAS without BSA. For CI analysis, CI was stimulated with pyruvate (10 mM), malate (0.5 mM), and ADP (4 mM); CI was inhibited with rotenone (1 μM); CII was stimulated by succinate (10 mM); and maximal respiration was stimulated with FCCP (1 μM). For CII analysis, CI was inhibited with rotenone (1 μM) and CII stimulated with succinate (10 mM) and ADP (4 mM); ATP-linked respiration was inhibited with oligomycin (1 μM); maximal respiration was stimulated by FCCP (1 μM); and respiration was decreased to non-mitochondrial levels with antimycin A (0.5 μM). At the end of the assay, normalization values for cell count were obtained as described above (“Normalization for Cell Count”), and OCR measurements were normalized against these values.

Heavy membrane isolations

Cells (~ 6 × 107 per sample) were treated as indicated for 24 h. Cells were then pelleted at 400 × g for 5 min at 4 °C and washed twice with 3 mL trehalose isolation buffer (TIB: 300 mM trehalose, 10 mM HEPES, 10 mM KCl, 1 mM EGTA, 0.1% BSA fraction V). Cells were resuspended in TIB supplemented with protease inhibitor and kept on ice. Cell suspensions were passed through a 27-gauge needle 13 times. The homogenate was centrifuged at 1,000 × g at 4 °C for 10 min, the supernatant collected, and centrifuged again at the same conditions to ensure that no unlysed cells or nuclei were present. The resulting supernatant was centrifuged at 10,000 × g at 4 °C for 10 min, and the resulting pellet collected as the heavy membrane isolate.

In-Gel mitochondrial complex extractions and analyses

Heavy membranes were resuspended in extraction butter (1 M 6-amino-hexanoic acid, 50 mM Bis–Tris HCl pH 7.0) and quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. 90 μg was solubilized with digitonin (final concentration 6%) on ice for 10 min, followed by centrifugation at 13,000 × g at 4 °C for 20 min. Supernatant was collected and glycerol added for a final concentration of 10%. 45 μg of sample/lane was resolved using Criterion TGX™ 4%-15% gels (Bio-Rad Laboratories, CA, USA) using native conditions at 50 V for about 4 h. For in-gel CI assays, gels were soaked in 30 mL of 5 mM Tris/HCl pH 7.4 containing NTB (30 mg) and 300 μL of 10 mg/mL NADH; for in-gel CII assays, gels were soaked in 30 mL of 5 mM Tris/HCl pH 7.4 containing NTB (30 mg), 600 μL of 1 M sodium succinate, and 24 μL of 250 mM phenazine methosulfate. All in-gel assays were performed for 1 h at room temperature. 15 μg of samples were resolved on a separate Criterion TGX™ 4%-15% gel under native conditions at 120 V and stained with Coomassie Blue as a loading control. Bands were quantified using (Fiji Is Just) ImageJ.

Mitochondrial mass and mitochondrial membrane potential analyses

Cells were plated at an initial concentration of 2 × 105 cells/mL and treated with 500 nM KL-11743 or an equal volume of DMSO for 24 h. Untreated parental cells were incubated without fluorescent staining or FCCP treatment for an unstained control. FCCP was used as a positive control for both membrane depolarization and accumulation of mitochondrial ROS for each cell line by treating with 10 μM FCCP before placing in the dark at 37 °C with 5% CO2 for 30 min. Cells were stained for 30 min using 200 nM MitoTracker™ Green FM or 100 nM TMRE, supplemented with DMSO, KL-11743, or FCCP, and placed in the dark at 37 °C with 5% CO2. Cells were collected in round-bottom polypropylene test tubes and centrifuged for 5 min at 450 × g at 4 °C. Supernatants were discarded and pellets were resuspended with 1 μg/mL DAPI in 300 μL of 1 × PBS, supplemented with DMSO, KL-11743, or FCCP. Unstained parental cells were resuspended in 300 μL of 1 × PBS without DAPI. All prepared stained cells and controls were analyzed with a BD FACSCanto II Clinical Flow Cytometry System with the parameters defined by the unstained and stained parental cells. All conventional flow cytometry data analysis was conducted through the FCS Express 7 computer software (version: 7.14.0020). Overall cell size was considered upon analysis of mitochondrial mass and mitochondrial membrane potential by flow cytometry as raw forward scatter-area (FSC-A) values for each cell was divided by the mean FSC-A of the population of interest to convert FSC-A values to a normalization factor centered on 1.00. Mitochondrial mass and mitochondrial membrane potential were determined by normalizing each cell’s mean fluorescent intensity (MFI) for MitoTracker™ Green FM or TMRE against the normalized FSC-A values.

Genomics analyses

DNA sequencing: AML patient’s peripheral blood mononuclear cells were employed for extracting genomic DNA using a DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions. DNA quality and quantity were assessed by Agilent 2100 Bioanalyzer system and Qubit (Thermo Fisher Scientific). DNA sequencing and mutational analyses were performed using the Ion Torrent platform and Ion Reporter v5.14; annotations were defined using the Oncomine Myeloid Assay Annotations v1.2 r.0, and using the following references: hg19, Oncomine Myeloid DNA Hotspots v1.3, Oncomine Myeloid DNA Mask – 318 – v1.2, Oncomine Myeloid DNA Regions – 318 – v1.0 (Thermo Fisher Scientific). Cell line RNA-Seq: NB4, and THP-1, RNA-seq scaled mRNA expression was performed using The Broad Institute’s Cancer Cell Line Encyclopedia (www.broadinstitute.org/ccle). iPSC RNA-Seq: Analysis was performed on the publicly available data GEO: GSE92494. The RNA read counts and metadata were obtained using the function “getGEO” from the R-package “GEOquery”. This data was later rearranged to construct a “DESeqDataSet” object containing only the samples of interest and genes with more than 10 reads. Principal component analysis was conducted using the function “plotPCA” on the regularized log transformation (“rlog” function of the R-package “DESeq2”) of normalized read counts. The differential expression analysis was performed with the R-package “DESeq2” comparing normal versus AML samples, yielding as significant those with an adjusted p-value (Benjamini & Hochberg) < 0.05 and log2FC > 2 or log2FC < -2. To generate the heatmap of 1460 differentially expressed genes, the read counts were scaled, log normalized and plotted using the R-package “ComplexHeatmap” with default row clustering. To visualize ontology pathways of interest, the significance cutoff was set at an adjusted p-value (Benjamini & Hochberg) < 1E-5 and log2FC > 1 or log2FC < -1. The differentially expressed data frame was filtered by each gene list downloaded from the Molecular Signatures Database (MSigDB) and graphed as volcano plots using Prism—GraphPad and Inkscape. The Reactome Pathway Database (Mitochondrial Protein Import and Translation), The Hallmark gene sets (Oxidative Phosphorylation and Glycolysis), and WikiPaths (Electron Transport Chain assembly) were utilized.