AML cell lines, and culture conditions

This study was conducted in accordance with the approved guidelines of the World Medical Association’s Declaration of Helsinki and the guidelines of the Ethics Committee of the Province of Salzburg. The AML cell lines MOLM-13, MV4-11 (both FLT3-ITD mutant), OCI-AML3 and THP-1 (both FLT3-wt; all purchased from Leibniz-Institut DSMZ GmbH) were cultured in RPMI-1640 medium (Sigma-Aldrich, Catalog number: R0883) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Catus Biotech, Catalog number: BS-2020-500), 1% penicillin and streptomycin (Sigma-Aldrich, Catalog number: P4333), and 2 mM L-glutamine (Sigma-Aldrich, Catalog number: G7513). PBMC’s were collected from fresh buffy coats of healthy donors via gradient density centrifugation using Histopaque®-1077 (Sigma-Aldrich, Catalog Number: 10771). ACK lysis buffer was used to remove red blood cells. PBMC’s were cultured in complete RPMI-1640 medium as described above. All cells were cultured at 37 °C, 5% CO2 in a humified atmosphere and regularly screened for mycoplasma contamination (MycoAlert™ PLUS Mycoplasma Detection Kit; Lonza, Catalog Number: LT07-705) following the manufacturer’s instructions.

2 × 105 cells/mL were seeded in appropriate cell culture plates and incubated for the indicated time points. CP-456773 (Sigma-Aldrich, Catalog number: PZ0280) was dissolved in sterile water, CY-09 (Tocris, Catalog number: 6436), Disulfiram (Selleckchem, Catalog number: S1680), Midostaurin (Sigma-Aldrich, Catalog number: M1323), Quizartinib (Selleckchem, Catalog number: S1526) and Bortezomib (MedChemExpress, Catalog number: HY-10227) were dissolved in DMSO.

Quantitative real time polymerase chain reaction (qRT-PCR)

Cell pellets of cultured cells were directly lysed in Tri Reagent® (Sigma-Aldrich, Catalog number: T9424) and RNA was isolated according to the manufacturer’s instructions. Briefly, chloroform was added, and the lysates were spun down. LPA (Sigma-Aldrich, Catalog number: 56575) was used as a carrier and mixed with the RNA-containing phase. Isopropanol was used to precipitate the RNA, and thereafter 70% ethanol was added for washing. RevertAid H Minus M-MulV reverse transcriptase (Thermo Fisher Scientific, Catalog number: EP0451) was used to generate complementary DNA (cDNA) in an iCycler Thermal Cycler (Bio-Rad). To determine gene expression levels by qRT-PCR, a Luna® Universal Probe qPCR Master Mix (New England BioLabs® Inc, Catalog number: M3003) was used and the amplification reactions were performed on a Rotorgene 3000 (Qiagen Instruments). To determine relative gene/mRNA expression, the large ribosomal protein P0 (RPLP0) was used as a reference gene. Relative mRNA expression (x) was calculated as x = 2−Δct, where Δct represents the difference between the threshold cycle (ct) of the target gene and the reference gene. Following primers (Sigma-Aldrich) were used: FLT3: forward 5’-ACCTCAAGTGCTCGCAGAAGCA-3’, reverse 5’-GTTAGCCTTTCTATTCCAGACTCC-3’; GLB1: forward 5’-CACTCCACAATCAAGACCGAAGC-3’, reverse 5’-CTGTGCTGCATAGGGTGAGTTG-3’; TP53: forward 5’-CCTCAGCATCTTATCCGAGTGG-3’, reverse 5’-TGGATGGTGGTACAGTCAGAGC-3’; CDKN1A: forward 5’-AGGTGGACCTGGAGACTCTCAG-3’, reverse 5’-TCCTCTTGGAGAAGATCAGCCG-3’; RPLP0: forward 5’-GGCACCATTGAAATCCTGAGTGATGTG-3’, reverse 5’-TTGCGGACACCCTCCAGGAAG-3’.

Western blot

Cell pellets were directly lysed in 80 µL of 2x Laemmli sample buffer (Bio-Rad, Catalog number: 1610737) supplemented with 5% β-mercaptoethanol (Sigma-Aldrich, Catalog number: M6250). The lysates were applied and separated on 4–12% NuPAGE Bis-Tris gradient gels (Life Technologies, Catalog number: NP0321) and then blotted onto a 0.45 μm nitrocellulose membrane (Bio-Rad, Catalog number: 1620115) using a semi-dry transfer system. 5% nonfat dry milk in 1x TBS containing 0.1% Tween 20 was used to block unspecific binding sites on the membrane. All antibodies were purchased from Cell Signaling and used according to the manufacturer’s instructions: CDK6 (3136), CDK4 (12790), p21 (2947), phosphorylated Rb (Ser780, 8190), total Rb (9309), FLT3 (3462), phosphorylated STAT5 (Tyr694, 9351), total STAT5 (9363), phosphorylated p44/42 MAPK (= ERK1/2, Thr202/Tyr204, 9106), total p44/42 (9102), β-actin (4970), HRP-linked anti-rabbit secondary antibody (7074) and HRP-linked anti-mouse secondary antibody (7076). A ChemiDox™ MP Imaging System was used for chemiluminescent detection (Thermo Fisher, Catalog number: 34580) of proteins. ImageJ software (NIH) was used for densiometric quantification of Western blots. Phosphorylated proteins were normalized to the respective total protein bands and total proteins were quantified relative to the loading control β-actin.

Flow cytometric analyses

The AML cell lines were seeded in 24- or 48-well plates at a density of 2 × 105 cells/mL and harvested according to manufacturer’s instructions for the chosen readout. Flow cytometric data were detected with a BD FACS Canto II and analyzed using FlowJo (FlowJo v10.7.1, BD Biosciences).

For cell cycle analysis, cells were seeded, treated as indicated and harvested after 24 and 48 h of incubation. The cells were stained with FxCycle™ PI/RNase Staining Solution according to the manufacturer´s instructions (Invitrogen, Catalog number: F10797). Viable cells were identified using the fixable viability dye eFluor™ 780 (Invitrogen, Catalog number: 65-0865-18).

Apoptosis of inhibitor treated cells was measured after 48 h of incubation using an Annexin V Apoptosis Detection Kit according to the manufacturer´s instructions (Invitrogen, Catalog number: 88-8006-74). Apoptotic cells are presented as the sum of the percentage of Annexin V+/7-AAD− and Annexin V+/7-AAD+ cells.

Synergy assay

2 × 105 cells/mL (2 × 104 cells/well) were seeded in flat bottom 96 well plates (Greiner, Catalog number: 655180) and treated with serial twofold dilutions of the drugs of interest. Untreated cells served as a positive control (= 100% viability) and cells treated with 10 µM Bortezomib (MedChemExpress, Catalog number: HY-10227) were used as a negative control (= 0% viability). 48 h post treatment, cell viability was measured using the CellTiter-Blue Cell Viability Assay (Promega, Catalog number: G8081) on the Infinite 200 PRO reader (Tecan) according to the manufacturer’s instructions. The data was normalized according to the positive and negative controls. The mean of three independent experiments was determined and the expected drug combination responses thereof were calculated based on the ZIP reference model using SynergyFinder. The interaction between two drugs can be defined as follows: a ZIP value of less than − 10 indicates antagonistic effects, a value between − 10 and + 10 indicates an additive effect, and a value greater than + 10 indicates synergistic effects [31].

Co-expression analysis of FLT3 and NLRP3 using the UCSC Xena browser

The data provided by the UCSC Xena browser (https://xenabrowser.net/) was used for the co-expression analysis of NLRP3 and FLT3 in more than 30 different cancer types using the TCGA Pan-Cancer dataset. In total, 12.839 samples were used for the analysis including 173 AML samples [30].

Proteomics Chemicals

Dithiothreitol (DTT, ≥ 99.5%), formic acid (FA, 98.0-100%), iodoacetamide (IAA, ≥ 99.0%), sodium dodecyl sulfate (SDS, ≥ 99.5%) and triethylammonium bicarbonate (TEAB, pH 8.5, 1 mol/L) were obtained from Sigma-Aldrich (Vienna, Austria). Acetonitrile (ACN, ≥ 99.9%) and methanol (MeOH, ≥ 99.9%) were obtained from VWR International (Vienna, Austria). Ammonia (25%) and ortho-phosphoric acid (85%) were purchased from Merck (Burlington, MA, USA). Trypsin (sequencing grade modified, porcine) was obtained from Promega (Madison, WI, USA). A MilliQ Integral 3 instrument (Millipore, Billerica, MA, USA) was used for deionization of water.

Cell culture

MOLM-13 cells were seeded in a 48-well plate at a density of 2 × 105/mL (1 × 105/500µL/well). Cells were treated with 75 and 125 µg/mL of CP-456,773 and incubated for 24 h at 37 °C and 5% CO2. Biological replicates were generated by conducting this treatment scheme on three consecutive days. Cells were washed thoroughly with PBS before conducting proteomics sample preparation.

Sample preparation

S-Trap mini columns (Protifi, Huntington, NY, USA) were employed according to the manufacturer´s instructions with minor adjustments: a cell pellet of approximately 1 × 106 cells was lysed in 5% SDS and 50 mmol/L TEAB (pH 7.55) at 95 °C for 5 min followed by sonication in a Bioruptor device (Diagenode, Liège, Belgium) for 10 min. After a centrifugation step, protein content was analyzed by a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Vienna, Austria). Denaturation and reduction of proteins were performed by supplementation of DTT to 40 mmol/L and incubation at 95 °C for 10 min. Reduced cysteine residues were alkylated by the addition of IAA to a concentration 80 mmol/L and incubation at 21 °C in the dark for 30 min. After a precipitation step and intense washing, 10 µg of trypsin were solubilized in 50 mmol/L TEAB (pH 8.5), added to the S-Trap matrix and incubated at 37 °C for 18 h. Peptides were eluted and subsequently dried at 30 °C using a vacuum centrifuge. Samples were resuspended in 100 mmol/L TEAB (pH 8.5) to a concentration of 1.0 mg/mL. 100 µg of peptides of each sample were labeled by a TMT 10plex™ kit (Thermo Fisher Scientific) according to the manufacturer´s instructions. Samples were labeled with the tags 126 to 130 C. All nine samples were pooled and dried at 30 °C in a vacuum centrifuge. The combined sample was resuspended in H2O + 20 mmol/L ammonium formate (pH 10.0) to a concentration of 1.8 mg/mL.

High-performance liquid chromatography and mass spectrometry

The pooled peptide sample was subjected to high-pH reversed phase fractionation without any prior purification. This off-line fractionation was carried out on an Agilent 1100 Series capillary LC system (Santa Clara, CA, USA). The peptides were separated on two sequentially linked Gemini NX-C18 columns (150 × 2.0 mm i.d., 3 μm particle diameter, 110 Å pore size) purchased from Phenomenex Inc. (Aschaffenburg, Germany) that were connected by a short 20 μm i.d. connective tubing. Mobile phases A (H2O + 20 mmol/L ammonium formate, pH10) and B ((90.0% ACN / 10.0% H2O) + 20 mmol/L ammonium formate, pH 10) were prepared according to Dwivedi et al. and Gilar et al. [58, 59]. A stepped linear gradient was applied: 1.0% B for 15.0 min, 1.0 − 30.0% B for 185.0 min, 30.0 − 60.0% for 30.0 min, 80.0% B for 30.0 min and 1.0% B for 40.0 min. The flow rate was set to 150 µL/min, the column oven temperature to 40 °C and 100.0 µl of sample were injected. 30 independent fractions were collected by an integrated automatic sample fraction collector system at uniform time slices starting at time point 12.0 min until time point 264.0 min. These 30 fractions were pooled into six fractions employing sample concatenation as reviewed by Yang et al. increasing orthogonality of the two chromatographic dimensions both relying on reversed phase separation principles [60]. Pooled fractions were dried at 45 °C in a vacuum centrifuge and upon dryness resuspended in 16 µl of H2O + 0.1% FA.

As a second dimension, acidic reversed phase HPLC was employed using a 2000 mm µPAC™ C18 column (PharmaFluidics, Ghent, Belgium). These nano scale chromatographic separations were carried out on a nanoHPLC instrument (UltiMate™ U3000 RSLCnano, Thermo Fisher Scientific, Germering, Germany) at a flow rate of 300 nL/min and a column oven temperature of 50 °C. Mobile phase solution A contained H2O + 0.10% FA and mobile phase B ACN + 0.10% FA. After solvent B was kept at 1.0% B for 5.0 min, a linear gradient to 40.0% B in 595.0 min was applied. This gradient was followed by a purging step at 90.0% B for 30.0 min. The column was re-equilibrated at 1.0% B for 100.0 min. 1.0 µl of each fraction was injected using a microliter pick-up mode (5.0 µl loop volume). Each fraction was measured once.

The nanoHPLC was hyphenated to a quadrupole-Orbitrap hybrid mass spectrometer (Thermo Scientific QExactive Plus benchtop quadrupole-Orbitrap mass spectrometer) via a Nanospray Flex ion source (both from Thermo Fisher Scientific, Bremen, Germany). The source was equipped with a SilicaTip emitter with 360 μm o.d., 20 μm i.d. and a tip i.d. of 10 μm purchased from New Objective (Woburn, MA, USA). The mass spectrometer was operated with following instrument settings: spray voltage of 1.5 kV, S-lens RF level of 55.0, capillary temperature of 320 °C and an MS1 AGC target of 3e6 in an m/z range of 400–2000 with a maximum injection time of 100 ms. A MS1 scan at a resolution setting of 70,000 at 200 m/z was followed by 15 data-dependent MS2 scans at a resolution of 35,000 at m/z 200. Target peptides were fragmented by HCD at 32.0 NCE in a 2.0 m/z isolation window with an AGC target of 1e5 and a maximum injection time of 100 ms. A dynamic exclusion setting of 30 s was applied. Pierce LTQ Velos ESI Positive Ion Calibration Solution from Life Technologies (Vienna, Austria) was used for calibration of the instrument.

Data evaluation

Acquired raw data was evaluated using MaxQuant software (v1.6.3.4) [61] in default settings correcting for isotope impurities in TMT reagents (provided by the manufacturer). Uniprot database entries including both Swiss-Prot as well as TrEMBL entries for homo sapiens (access: 10.03.2019) were provided for MaxQuant protein identification [62]. Obtained protein groups were further processed using Perseus (v1.6.6.1): Protein groups were filtered removing potential contaminants, proteins that were only identified by site and reverse sequence matches [63]. Only those protein groups providing quantitative values for all nine reporter ion channels were further processed, including log2-transformation and normalization by subtraction of the median. For statistical analysis and data representation, the R software version 3.6.1 as well as GraphPad Prism version 8.0.2. (GraphPad Software, San Diego, CA, USA) were employed [64].

Transmission electron microscopic (TEM) preparation and visualization

MOLM-13 cells were either left untreated or treated with 125 µg/mL of CP-456773 for 48 h and then processed for TEM imaging as described previously [21]. The cells were high-pressure frozen (HPF), cryosubstituted and then embedded in epoxy resin. Thereafter, the samples were trimmed and cut to ultrathin sections (~ 70 nm). Samples on Formvar-coated copper grids were then transferred to the transmission electron microscope (TEM) and images were recorded on a LEO 912 AB Omega TEM at 80 kV. Digital images were recorded with a bottom-mounted 2 K CCD camera from Tröndle TRS Sharp Eye (Tröndle, Moorenweis, Germany). All TEM images were filtered at zero energy loss. iTEM 5.0 software (Olympus SIS, Münster, Germany) was used for TEM implementation and the recording process.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). Data was tested for normality and appropriate statistical tests were used: Statistical analyses were performed by a paired t-test for the analysis between two groups, one-way ANOVA with Tukey’s or Šídák’s post-hoc test or a two-way ANOVA with Šídák’s post hoc test for multiple comparisons. Significance levels are defined as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant.