The anti-leukemia drug ponatinib attenuates NLRP3 inflammasome activation in macrophages

Abstract

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is an established driver of inflammation in diseases such as diabetes, Alzheimer's disease, and gout. Previously, we screened 875 FDA-approved drugs for NLRP3 inhibitors and identified ponatinib as one of the five candidates that reduced NLRP3 inflammasome activation without causing cytotoxic effects in bone marrow-derived macrophages (BMDMs). Therefore, we hypothesize that ponatinib may be an effective NLRP3 inflammasome inhibitor. We performed dose curves and cytotoxicity assays to determine an effective in vitro concentration of ponatinib in BMDMs (1 μM) and primary microglia (0.5 μM) that reduced IL-1β secretion without inducing cytotoxicity. In BMDMs, ponatinib inhibited Nlrp3- and Caspase-1-dependent IL-1β and IL-18 secretion and significantly reduced caspase-1 processing. Ponatinib also reduced IL-1β secretion by microglia, indicating possible NLRP3 inflammasome inhibition in this cell type, but further testing is required. Although ponatinib has previously been demonstrated to drive cardiotoxicity-mediated inflammation, these data suggest that at specific doses, in vitro ponatinib can be non-cytotoxic and effective in attenuating NLRP3 inflammasome activation in macrophages and microglia.

1. Introduction

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex that regulates innate immune signaling (Lamkanfi and Dixit, 2014). NLRP3 activation requires two steps: 1) priming and 2) activation (Lamkanfi and Dixit, 2014). Priming occurs through the triggering of pattern recognition receptors (PRRs) or other receptors that prompt the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to upregulate the expression of pro-IL-1β and pro-IL-18 (Lamkanfi and Dixit, 2014). An activating signal then triggers inflammasome assembly and recruitment of pro-caspase-1 by the CARD domain in NLRP3's adaptor, apoptosis-associated speck-like protein containing a CARD (ASC). Once recruited, pro-caspase is cleaved and activated. Active caspase-1 then cleaves pro-IL-1β and pro-IL-18 into their proteolytic forms which are released from the cell (Lamkanfi and Dixit, 2014). In addition to the release of IL-1β and IL-18, NLRP3 activation results in gasdermin-D dependent pyroptosis (Lamkanfi and Dixit, 2014). While NLRP3 activation is a crucial component of innate immunity, its overactivity drives inflammation in several diseases, including diabetes, atherosclerosis, and Alzheimer's disease (Swanson et al., 2019). Therefore, there is a pressing need to identify novel inhibitors of the NLRP3 inflammasome.

To address this need, our lab acquired a library of 875 FDA-approved drugs from Cayman Chemical Company (Cat. No. 23538) and screened them for NLRP3 inhibitors using bone marrow derived macrophages (BMDMs) (Shippy and Ulland, 2025; Shippy et al., 2026). Through this screen, we identified five drugs, including ponatinib, as inhibitors of the NLRP3 inflammasome (Shippy and Ulland, 2025). Here, we detail the NLRP3 inhibitory effects of ponatinib, an FDA-approved multi-target tyrosine kinase inhibitor (TKI) used for the treatment of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL), as an NLRP3 inhibitory compound in BMDMs. Utilizing dose curves and cytotoxicity/viability assays, we identified an effective dose at which ponatinib decreases NLRP3 activation in BMDMs and primary microglia without inducing cytotoxicity or affecting viability. Furthermore, we found that ponatinib decreases IL-1α, IL-1β, and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion in BMDMs but does not affect secretion of several other cytokines and chemokines (IL-6, IL-10, TNF-α, MIP-1α, RANTES, CXCL1, MDC, and TARC). Overall, our work shows that ponatinib effectively inhibits NLRP3 activation in BMDMs and primary microglia.

2. Methods

2.1. Mice

C57BL/6 J (Cat. No. 000664), Casp1^−/− (Cat. No. 016621) (Kuida et al., 1995) and Nlrp3^−/− (Cat. No. 021302) (Kovarova et al., 2012) mice were purchased from The Jackson Laboratory. Casp1^−/− and Nlrp3^−/− mice were backcrossed onto the C57BL/6 J background for an additional five generations before use. All mice were bred and housed in specific-pathogen-free conditions under a 12 h light/dark cycle. Drinking water and food were available ad libitum.

2.2. Primary cell culture

BMDM were prepared as previously described (Shippy et al., 2020). Before use in experiments, BMDM were cultured in RPMI 1640 (Gibco, Cat. No. 11875–093) with penicillin/streptomycin (1×), Glutamax (1×) (Gibco, Cat. No. 35050–061), sodium pyruvate (1×) (Gibco, Cat. No. 11360–070), non-essential amino acids (1×) (Gibco, Cat. No. 11140–050),10% heat-inactivated fetal bovine serum (FBS) (Cytvia HyClone, Cat. No. SH30071.03HI) and 20% L-cell conditioned medium for 5 days.

Primary microglia were prepared from female wild-type C57BL/6 J mice (The Jackson Laboratory) as previously described (Shippy et al., 2022). Microglia were isolated using Magnetic Activated Cell Sorting (MACS, Miltenyi Biotec) according to manufacturer's instructions. Briefly, mice were perfused with cold PBS containing heparin (0.1%). Brains were collected in C-tubes (Miltenyi Biotec, Cat. No. 130–096-334) and dissociated using a Neural Tissue Dissociation Kit (Miltenyi Biotec, Cat. No. 130–093-231). Microglia were labeled with anti-mouse CD45 magnetic beads (Miltenyi Biotec, Cat. No. 130–052-301) and isolated on LS columns (Miltenyi Biotec, Cat. No. 130–042-401). Approximately 40,000 microglia/well were seeded on poly-l-lysine coated 48-well plates in complete RPMI media supplemented with LCCM (20%) and human TGF-β (10 ng/ml) (PeproTech, Cat. No. 100–21). Primary microglia were incubated for 3 days before the media was replaced with complete RMPI supplemented with LCCM (20%). Cells were then incubated for an additional 7 days before use in assays.

2.3. Dose curves

For BMDM dose curves, ponatinib was prepared at concentrations of 4 μM, 2 μM, 1 μM, 0.5 μM, and 0.25 μM to determine an effective in vitro concentration. After differentiation, BMDM (400,000 cells/well) were cultured in 24-well plates. Media for this experiment consisted of glucose-free DMEM (Gibco, Cat. No. 11966–025) supplemented with glucose (3 mM) (Gibco, Cat. No. A24940–01) and recombinant murine macrophage colony stimulating factor (MCSF) (10 ng/ml) (PeproTech, Cat. No. 315–02) (plating media). To prime the inflammasome, all cells were treated with lipopolysaccharide (LPS) (50 ng/ml) from Escherichia coli O26:B6 (Sigma, Cat. No. L2654) for 4 h at 37 °C, 5% CO₂ before incubation with either the different ponatinib doses or dimethyl sulfoxide (DMSO) (control) for 15 min. To activate NLRP3, all BMDM were then treated with adenosine triphosphate (ATP) (5 mM) (Research Products International, Cat. No. A300305) for 1 h. The release of IL-1β into supernatants was detected by enzyme-linked immunosorbent assay (ELISA) as described below. Three independent biological replications of this experiment were performed.

For microglia dose curves, ponatinib was prepared at concentrations of 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, and 0.06 μM to determine an effective in vitro drug concentration for IL-1β inhibition in microglia. Primary microglia were isolated and plated on poly-l-lysine coated 48-well plates (40,000 cells/well) in complete RPMI media supplemented with LCCM (20%) and all groups were stimulated with LPS (50 ng/ml) for 3.5 h at 37 °C, 5% CO₂. Microglia were then incubated with the different ponatinib concentrations or DMSO (control) for 15 min followed by stimulation of all groups with ATP (5 mM) for 1 h. The release of IL-1β into supernatants was detected by enzyme-linked immunosorbent assay (ELISA) as described below. Three independent biological replications of this experiment were performed.

2.4. Cytotoxicity assays

We then assessed the toxicity of any dose that resulted in a significant reduction in IL-1β production relative to the DMSO control. BMDM (20,000 cells/well) were cultured in 96-well tissue culture plates and allowed to incubate overnight. Media utilized in this experiment consisted of phenol red-free RPMI 1640 (Gibco, Cat. No. 11835–030) with 5% heat-inactivated FBS and MCSF (10 ng/ml). BMDMs were treated with the doses of interest or relevant controls for 15 min, 6 h, or 24 h at 37 °C, 5% CO₂. Three independent biological replications of this experiment were performed.

Primary microglia were isolated and plated on poly-L-lysine coated 48-well plates (40,000 cells/well) in phenol red-free RPMI 1640 supplemented with 5% heat-inactivated FBS and MCSF (20 ng/ml). Cells were incubated with media, drug, and DMSO for 1.25, 6, or 24 h at 37 °C, 5% CO₂. Three independent biological replications of this experiment were performed.

Cytotoxicity was measured by release of lactate dehydrogenase (LDH) after treatment with ponatinib using the Cytotox 96^® Non-Radioactive Cytotoxicity Assay (Promega, Cat. No. G1780) following manufacturer's instructions. Any dose that significantly (P < 0.05) increased LDH release relative to the DMSO control was considered unsafe. The effective in vitro concentration of ponatinib was determined using the highest drug concentration that significantly reduced IL-1β secretion without being toxic to cells.

2.5. Cell viability assays

A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine cell viability at 1 μM ponatinib. Because LDH has a half-life of nine hours, this assay was included to have a secondary measure of cell cytotoxicity/viability. The cell viability assay was performed using the MTT Cell Proliferation Assay Kit (Abcam, Cat. No. Ab211091) as previously described (Oliai et al., 2024; Wang et al., 2022). Briefly, BMDM were seeded in a 24-well plate (250,000 cells/well) in complete RPMI media supplemented with LCCM (20%) and incubated overnight at 37 °C, 5% CO₂. The following day, the media was aspirated and replaced with phenol red-free RPMI 1640 supplemented with heat-inactivated FBS (10%) and MCSF (10 ng/ml). Cells were incubated with media (negative control), DMSO, and ponatinib (1 μM) for 1.25, 6 and 24 h at 37 °C, 5% CO₂. Following incubation, the media was aspirated and replaced with serum-free RPMI 1640 (0.2 ml/well) and MTT reagent (0.2 ml/well) and incubated for 3 h at 37 °C, 5% CO₂. A MTT background control was used with cell-free wells. After incubation, MTT solvent (0.3 ml/well) was added to each well and incubated for 15 min at room temperature on a shaking platform before absorbance was read at 590 nm. Percent cell viability was calculated by subtracting the MTT background control average from the value of the experimental samples, then dividing by the negative control average and multiplying the quotient by 100.

2.6. NLRP3 inflammasome assays

To assess the effect of ponatinib (Cat. No. 11494) on NLRP3 activation in BMDMs, the optimal in vitro concentration of ponatinib (1 μM), determined by the dose curve, cytotoxicity and viability assays described above, was utilized in the following experiments. Wild type (WT), Casp1^−/−, and Nlrp3^−/− BMDMs were cultured and treated as described above, but with the appropriate controls for all treatment conditions. Additionally, cells were treated either with ponatinib (1 μM) or DMSO. Following treatment, supernatants were collected and stored at −80 °C until use. Five independent biological replications of this experiment were performed.

Ponatinib was used at a concentration of 0.5 μM in microglia assays to determine its ability to inhibit the NLRP3 inflammasome. Primary microglia were isolated and plated on poly-l-lysine coated 48-well plates (40,000 cells/well) in complete RPMI media supplemented with LCCM (20%). Microglia were incubated with LPS, ponatinib (0.5 μM)/DMSO, and ATP as described above. Following treatment, supernatants were collected and stored at −80 °C until use. Three independent biological replications of this experiment were performed.

2.7. Caspase-Glo^® 1 inflammasome assay

To determine the effect of ponatinib (1 μM) on caspase-1 activity in WT BMDM, we utilized the Caspase-Glo^® 1 Inflammasome Assay (Promega, Cat. No. G9951) to detect processed caspase-1 in cell supernatants as previously described (Shippy and Ulland, 2025). Three independent biological replications of this experiment were performed.

2.8. Inflammation assays

To determine if ponatinib (1 μM) affects inflammatory pathways other than the NLRP3 inflammasome, we cultured WT BMDMs (400,000 cells/well) in a 24-well plate and treated with ponatinib (1 μM)/DMSO for 15 min before treatment with LPS (50 ng/ml) for 6 h. Supernatants were collected and stored at −80 °C until use. Three independent biological replications of this experiment were performed.

2.9. ELISAs

IL-1β in WT BMDM dose curve supernatants was detected using the Mouse IL-1β/IL-1F2 DuoSet ELISA (R&D Systems, Cat. No. DY401) following manufacturer's instructions.

Secreted IL-1β and IL-18 in WT BMDM NLRP3 inflammasome experiment supernatants was detected using the Mouse IL-1β/IL-1F2 DuoSet ELISA (R&D Systems, Cat. No. DY401) and IL-18 using the Mouse IL-18 DuoSet ELISA (R&D Systems, Cat. No. DY7625) following manufacturer's instructions. Additionally, IL-1α, IL-1β, IL-6, IL-10, TNF-α, MIP-1α, RANTES, GM-CSF, CXCL1, MDC, and TARC were detected using a multiplex (11-plex) assay (Quansys Biosciences).

IL-6, TNF-α, MCP-1, CXCL-1, RANTES, and MIP-1α were detected in WT BMDM supernatants from the inflammation cell culture experiments using the Mouse IL-6 DuoSet ELISA (R&D Systems, Cat. No. DY406), Mouse TNF-α DuoSet ELISA (R&D Systems, Cat. No. DY410), Mouse CCL2/JE/MCP-1 DuoSet ELISA (R&D Systems, Cat. No. DY479), Mouse CXCL1/KC DuoSet ELISA (R&D Systems, Cat. No. DY453), Mouse CCL5/RANTES DuoSet ELISA (R&D Systems, Cat. No. DY478), and Mouse CCL3/MIP-1α (R&D Systems, Cat. No. DY450), respectively, following manufacturer's instructions.

Primary microglia supernatants from the dose curves and NLRP3 inflammasome cell culture experiments were assayed for IL-1β using the Mouse IL-1β/IL-1F2 DuoSet ELISA (R&D Systems, Cat. No. DY401) following manufacturer's instructions.

2.10. Statistical assays

Statistical analyses were performed using Prism (GraphPad, v.10.0.2). Data are presented as mean ± standard error of the mean (SEM). Comparison between two groups was performed using a Student's t-test. Comparison between multiple groups was performed using one-way analysis of variance (ANOVA) with a Dunnett's multiple comparisons test and two-way ANOVA with a Tukey's multiple comparisons test. A P-value <0.05 (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) was used as the significance cutoff.

3. Results

3.1. Ponatinib Inhibits NLRP3 in BMDMs

To identify drugs that inhibit the NLRP3 inflammasome, we purchased a library of 875 FDA-approved drugs from Cayman Chemical Company (Cat. No. 23538) and screened them for compounds capable of decreasing IL-1β secretion in BMDMs (Shippy and Ulland, 2025; Shippy et al., 2026). Ponatinib reduced the secretion of IL-1β in BMDMs by 37% in our primary screen and significantly decreased IL-1β secretion in our secondary screen (Fig. S1). To identify a working concentration of ponatinib in BMDMs, we performed a dose curve and quantified IL-1β secretion by ELISA after treatment with ponatinib at 4 μM, 2 μM, 1 μM, 0.5 μM, and 0.25 μM. Ponatinib significantly reduced IL-1β secretion at all doses except 0.25 μM (Fig. 1A). To confirm this was due to inhibition of the NLRP3 inflammasome rather than toxicity or loss of viability, we assessed cytotoxicity after treatment with ponatinib at 15 min, 6 h, or 24 h. We quantified the release of LDH as a metric of cell death and found ponatinib did not increase cytotoxicity relative to DMSO (the vehicle control) at 1 μM and 0.5 μM (Fig. 1B). Cell viability was assessed via quantification of MTT after treatment with ponatinib (1 μM) for 1.25 h, 6 h, and 24 h. We found ponatinib (1 μM) decreased cell viability significantly at 24 h, but not at 6 h or 1.25 h (Fig. S2). Because 1 μM decreased IL-1β most significantly without inducing cytotoxicity, we utilized this concentration in all future BMDM experiments.

Fig. 1.

Ponatinib attenuates NLRP3 inflammasome activation in BMDMs. An effective in vitro dose of ponatinib in BMDMs was determined using dose curves (all groups were treated with LPS and ATP) (A) and cytotoxicity assays (B). Secretion of IL-1β (C) and IL-18 (D) in supernatants from WT, Casp1^−/−, and Nlrp3^−/− BMDMs were determined by ELISA. (E) Caspase-1 levels were determined in WT BMDM supernatants using a bioluminescent assay. Comparisons in panel A–B were performed one-way ANOVA with a Dunnett's multiple comparisons test. Comparisons in panels C–E were performed using two-way ANOVA with a Tukey's multiple comparisons test. Data is presented as mean ± SEM. ****P < 0.0001, ***P < 0.001, ns = not significant.

To further characterize the effect of ponatinib (1 μM) on NLRP3 inhibition, we quantified IL-1β and IL-18 production in the presence and absence of NLRP3 priming (LPS) and activating (ATP) factors in WT BMDMs. As expected, BMDMs secreted both IL-1β and IL-18 only in response to stimulation with both LPS and ATP. Secretion of both cytokines was inhibited by ponatinib (Fig. 1C and D). Caspase-1^−/− and Nlrp3^−/− BMDMs did not secrete IL-1β or IL-18 in response to stimulation with LPS, ATP, or both, confirming that IL-1β and IL-18 produced in our experiments were both caspase-1 and NLRP3 dependent (Fig. 1C and D). We then quantified caspase-1 activity in cell supernatants utilizing the Caspase-Glo^® 1 Inflammasome Assay and found ponatinib significantly reduced caspase-1 activity in LPS primed and ATP activated WT BMDMs (Fig. 1E). These experiments confirmed ponatinib inhibits the NLRP3 inflammasome in BMDMs. We then sought to determine if ponatinib reduces the release of other cytokines and chemokines. Secretion of several cytokines and chemokines (IL-1α, IL-1β, GM-CSF, IL-6, IL-10, TNF-α, MIP-1α, RANTES, CXCL1, MDC, and TARC) by BMDMs treated for NLRP3 activation were quantified using a multiplex (11-plex) assay (Quansys Biosciences). Ponatinib treatment significantly decreased the secretion of IL-1α, IL-1β, and GM-CSF (Fig. 2A-C).

Fig. 2.

Anti-inflammatory properties of ponatinib in NLRP3 activated BMDMs. WT BMDMs were stimulated with LPS (50 ng/mL) for 4 h, then ponatinib (1 μM) or DMSO for 15 min prior to activation with ATP (5 mM) for 1 h. Supernatants were assayed for several cytokines/chemokines by ELISA. Comparisons were performed using two-way ANOVA with a Tukey's multiple comparisons test. Data is presented as mean ± SEM. ****P < 0.0001, ns = not significant.

In a separate experiment designed to assess ponatinib's effect on general inflammation, BMDMs were treated with ponatinib (1 μM) for 15 min before stimulation with LPS (50 ng/mL) for 6 h. We quantified the release of CXCL1, IL-6, RANTES, TNF-α, MIP-1α, and MCP-1 by ELISA and observed a significant decrease in all six proteins (Fig. S3). However, we observed a trend toward decreased viability at 6 h of treatment with ponatinib (Fig. S2B) and determined any decreases in cytokine production in the general inflammation assay could not be solely attributed to the inhibition of inflammatory pathways. Therefore, we concluded that ponatinib (1 μM) inhibits NLRP3 activation (Fig. 1C-E) but is not generally anti-inflammatory.

3.2. Ponatinib decreases IL-1β Secretion in primary microglia

To determine if ponatinib inhibits the NLRP3 inflammasome in microglia as in BMDMs, we performed a dose curve treating primary microglia with doses at 1 μM, 0.5 μM, 0.25 μM, 0.13 μM, and 0.06 μM under the same conditions used for the BMDM dose curve. IL-1β was detected by ELISA. We found that all doses significantly reduced IL-1β secretion in primary microglia (Fig. 3A). To confirm that this reduction was not due to cytotoxicity, LDH release was measured after treatment with ponatinib for 1.25 h, 6 h, and 24 h. We tested cytotoxicity at 1 μM, 0.5 μM, 0.25 μM and found ponatinib induced cytotoxicity at 1 μM, but not 0.5 µM or 0.25 μM (Fig. 3B; Fig. S4). Therefore, 0.5 μM was utilized in all following microglia experiments, as it most effectively decreased IL-1β secretion without cytotoxic effects. We tested the effects of ponatinib (0.5 μM) on microglia in the presence or absence of both LPS and ATP and found ponatinib significantly reduced IL-1β secretion from primary microglia at 0.5 μM (Fig. 3C).

Fig. 3.

Ponatinib attenuates NLRP3 inflammasome activation in primary microglia. An effective in vitro dose of ponatinib in primary microglia was determined using dose curves (all groups were treated with LPS and ATP) (A) and cytotoxicity assays (B). Secretion of IL-1β (C) in supernatants from primary microglia using the optimal in vitro dose (0.5 μM) of ponatinib was determined by ELISA. Comparisons in panel A were performed one-way ANOVA with a Dunnett's multiple comparisons test. Comparisons in panel B were performed using a Student's t-test. Comparisons in panel C were performed using two-way ANOVA with a Tukey's multiple comparisons test. Data is presented as mean ± SEM. ****P < 0.0001, ***P < 0.001, ns = not significant.

4. Discussion

After screening 875 FDA-approved drugs for inhibitors of the NLRP3 inflammasome (Shippy and Ulland, 2025; Shippy et al., 2026), we identified ponatinib as a promising candidate. Through further experiments, we showed ponatinib decreases NLRP3 activation in BMDMs at a concentration of 1 μM without inducing measurable cytotoxicity or decreasing cell viability (Fig. 1; Fig. S2). Additionally, treatment with ponatinib at 1 μM decreased IL-1α and GM-CSF secretion by BMDMs (Fig. 2). In primary microglia, we found that ponatinib decreased IL-1β secretion at a dose of 0.5 μM without causing cytotoxicity (Fig. 3). However, we observed treatment with ponatinib at higher concentrations or longer duration resulted in increased cytotoxicity and decreased cell viability in both BMDMs and microglia (Fig. 1; Fig. S2; Fig. S4), indicating that ponatinib may be effective at specific concentrations before becoming cytotoxic. While the literature on ponatinib is divided regarding its effects on inflammation, our findings are in line with several studies reporting its anti-inflammatory effects (Lin et al., 2022; Chen et al., 2019; Wong et al., 2013; Wong et al., 2014).

It is well documented that treatment with ponatinib comes with the risk of severe cardiovascular side effects (Cortes et al., 2013); however, the specific mechanism of ponatinib's cardiotoxicity is not fully known. One study by Arzuk et al. found treatment with ponatinib contributed to oxidative stress leading to increased NLRP3 mRNA expression and IL-1β production in AML12 mouse hepatocytes (Arzuk, 2024). Notably, the concentrations of ponatinib utilized in this study were up to 100× that of our own, and cells were treated for much longer. This contrast in methodology may explain the difference in conclusions drawn between our studies. One mouse study by Tousif et al. found treatment with ponatinib led to increased S100A8/A9 alarmin production, triggering increased inflammation, including NLRP3 activation, and subsequent cardiotoxicity (Tousif et al., 2023). However, while this study found a robust inflammatory response to treatment with ponatinib, the authors did not fully differentiate between general systemic inflammation and NLRP3 activation induced by ponatinib in vivo. Still, the discrepancies between these studies (Arzuk, 2024; Tousif et al., 2023) and our own suggest ponatinib may have differing effects on inflammation and cell viability depending on the concentration, treatment duration, and cell type within the treatment model.

Other studies report that ponatinib has anti-inflammatory effects. Lin et al. found ponatinib reduced inflammation in leptin-deficient obese mice; ponatinib-treated mice showed significantly decreased macrophage infiltration and expression of IL-1β, IL-18, TNF-α, and IL-6 mRNA in adipose tissue (Lin et al., 2022). Also in this study, in vitro experiments examining free fatty acid induced inflammation showed treatment with ponatinib decreased IL-1β, IL-18, TNF-α, and IL-6 mRNA expression in BMDMs relative to controls (Lin et al., 2022). Similarly, Chen et al. found that treatment of influenza infected BALB/c mice with ponatinib was protective and significantly decreased inflammatory cytokines in bronchoalveolar lavage fluids (Chen et al., 2019). Furthermore, two studies by Wong et al. in 2013 and 2014 found treatment with ponatinib decreased doxorubicin induced IL-1β, IL-6, and CXCL1 gene expression and cytokine release both in mice and cultured BMDMs (Wong et al., 2013; Wong et al., 2014). Taken together with our study, this work suggests certain concentrations of ponatinib may be utilized to safely and effectively inhibit NLRP3 activation. However, future work is needed to understand why ponatinib may promote a pro-inflammatory response under different conditions.

To determine if ponatinib affects release of other cytokines and chemokines, we utilized a multiplex (11-plex) assay (Quansys Bio-sciences) to quantify the secretion of IL-1α, IL-1β, GM-CSF, IL-6, IL-10, TNF-α, MIP-1α, RANTES, CXCL1, MDC, and TARC from BMDMs treated for NLRP3 activation. As expected, treatment with ponatinib resulted in decreased IL-1β production (Fig. 2B). In addition, ponatinib significantly decreased IL-1α and GM-CSF release (Fig. 2A; Fig. 2C). All other cytokines remained unchanged (Fig. 2D-K). Interestingly, while IL-1β and IL-1α bind the same receptor (IL-1R) and exert similar effects, their production mechanisms differ (Eislmayr et al., 2022). The production of both IL-1β and IL-1α require the activation of NF-κB; however, IL-1α is active in its precursor form, unlike IL-1β, which requires processing by the inflammasome prior to secretion (Di Paolo and Shayakhmetov, 2016). However, secretion of IL-1α is in part facilitated by pyroptosis induced by NLRP3 activation (Di Paolo and Shayakhmetov, 2016). Therefore, the observed reduction in IL-1α secretion may be explained by ponatinib's inhibition of NLRP3. GM-CSF may have been decreased due to the reduction of IL-1β and IL-1α resulting in decreased IL-1R signaling and NF-κB activation (Schreck and Baeuerle, 1990). Our work suggests that ponatinib inhibits NLRP3 activation in BMDMs but does not significantly alter the secretion of NLRP3-independent cytokines and chemokines.

It is worth noting that while we tested the effect of ponatinib on ATP-dependent NLRP3 activation, there are other methods of NLRP3 activation. Specifically, other activating stimuli (e.g. monosodium urate, alum, nigericin, PM_2.5) (Caceres et al., 2024; Kelley et al., 2019) can be utilized to promote activation of the NLPR3 inflammasome in macrophages. Future work should focus on these alternate mechanisms of activation in addition to understanding by what mechanism ponatinib inhibits NLRP3. Ponatinib has been found to phosphorylate more than 100 kinases (O'Hare et al., 2009), some of which are directly related to NLRP3 inflammasome activation. Specifically, ponatinib has been shown to inhibit Janus kinase 2 (JAK2) (Liu et al., 2019) and signal transducer and activation of transcription 3 (STAT3) (Tan et al., 2018) which are both known drivers of NLRP3 inflammasome activation (Zhu et al., 2021; Luo et al., 2024). Further study of ponatinib's effect on NLRP3 activation should investigate the phosphorylation of these kinases as ponatinib's potential mechanism of inhibition.

This work presents ponatinib as an inhibitor of the NLRP3 inflammasome in murine BMDMs and primary microglia and is in line with a body of work suggesting ponatinib has anti-inflammatory effects. Future work to develop ponatinib into a safe anti-inflammatory agent should explore the potential mechanism by which ponatinib inhibits NLRP3 in these cell types and explore options for reducing cardiotoxic side effects.

Abbreviations:

NLRP3

NOD-like receptor family pyrin domain containing 3

BMDM

bone marrow derived macrophage

PRR

pattern recognition receptor

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

ASC

apoptosis-associated speck-like protein containing a CARD

TKI

tyrosine kinase inhibitor

CML

chronic myelogenous leukemia

Ph + ALL

Philadephia positive acute lymphoblastic leukemia

GM-CSF

granulocyte macrophage colony stimulating factor

LCCM

L-cell conditioned medium

MCSF

macrophage colony stimulating factor

LPS

lipopolysaccharide

DMSO

dimethyl sulfoxide

ATP

adenosine triphosphate

ELISA

enzyme-linked immunosorbent assay

LDH

lactate dehydrogenase

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

JAK2

Janus kinase 2

STAT3

signal transducer and activator of transcription 3

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.imbio.2026.153241.

Data availability

Data will be made available on request.