Therapeutic activation of PPARα contributes to NOTCH1 inhibition in T-cell acute lymphoblastic leukemia

Abstract

Background

The aberrant activation of the NOTCH1 signaling pathway underlies the aggressive malignancy and poor prognosis of T-cell acute lymphoblastic leukemia (T-ALL).

Methods

T-ALL cell lines (Jurkat and Molt4) were treated with chiglitazar to evaluate viability, proliferation, apoptosis, and cell cycle. RNA-seq, qRT-PCR, and Western blotting were used to examine NOTCH1 signaling. Mechanistic assays included luciferase reporter, DNA affinity precipitation, co-immunoprecipitation, and ChIP. In vivo, cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models were generated by intravenous engraftment of leukemic cells into sublethally irradiated mice, followed by treatment with chiglitazar alone or combined with venetoclax. Therapeutic efficacy was assessed by survival, flow cytometric tumor burden, and histopathology (HE and IHC).

Results

We report that therapeutic activation of peroxisome proliferator-activated receptor α (PPARα) significantly represses the leukemogenesis of T-ALL in vitro and in vivo by blocking the NOTCH1 signaling pathway. Mechanistically, PPARα directly binds to the promoter region of the NOTCH1 gene and inhibits its transcriptional activity. Furthermore, PPARα interacts with signal transducer and activator of transcription 3 (STAT3) and attenuates the transcriptional activation effect of STAT3 on the NOTCH1 gene promoter. Importantly, we also found that therapeutic activation of PPARα using chiglitazar synergizes with venetoclax to suppress T-ALL progression in PDX models.

Conclusions

We conclude that targeting PPARα to suppress T-ALL progression by blocking the NOTCH1 pathway represents a potential novel therapeutic strategy for the treatment of T-ALL.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12916-026-05045-x.

Background

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy arising from the malignant transformation of T-cell progenitors, characterized by high relapse rates and poor prognosis, particularly in adults and patients with refractory disease [1–3]. Although intensive chemotherapy regimens have improved survival rates, the toxicity-related mortality and limited salvage options for relapsed cases remain significant clinical challenges [4–6].

The molecular landscape of T-ALL is dominated by aberrant activation of the NOTCH1 signaling pathway, with activating mutations identified in over 65% of patients, driving constitutive leukemic cell proliferation, survival, and metabolic reprogramming [7, 8]. Consequently, pharmacological blockade of NOTCH1, particularly via γ-secretase inhibitors (GSIs), has been extensively pursued as a targeted therapy. However, the clinical translation of GSIs has been severely hampered by “on-target” gastrointestinal toxicity due to concurrent NOTCH2 inhibition in the intestinal epithelium and the rapid emergence of drug resistance [9, 10].

Emerging evidence suggests that this resistance is often mediated by compensatory signaling networks, with JAK-STAT signaling members playing pivotal roles. Constitutively activated STAT signaling has been shown to cooperate with oncogenic drivers to upregulate anti-apoptotic factors, such as Mcl-1 and Bcl-2, thereby sustaining leukemia cell survival under NOTCH1 inhibition [11–13]. Furthermore, recent studies indicate that the interplay between NOTCH1 and STAT3 orchestrates a complex metabolic-transcriptional network that fuels drug resistance in various malignancies [14]. Therefore, identifying novel regulatory mechanisms that can simultaneously disrupt NOTCH1 and STAT3 oncogenic dependencies without incurring unacceptable toxicity represents an urgent unmet need.

PPARα, a ligand-activated nuclear transcription factor, is canonically recognized for its central role in regulating lipid metabolism and energy homeostasis [15]. However, recent high-impact studies have unveiled a “non-canonical” function of PPARα as a potent tumor suppressor in various cancers [16], including hematologic malignancies. For instance, recent research demonstrated that therapeutic activation of PPARα significantly suppresses acute myeloid leukemia (AML) progression by disrupting the HIF1α-mediated metabolic axis and targeting leukemia stem cells [17]. More recently, it has been shown that co-targeting Bcl-2 inhibition alongside PPARα activation synergistically eradicates leukemic stem cell-like cells in AML [18], while combining an HDAC inhibitor with a PPAR agonist potently induces ferroptosis in these primitive populations [19]. These findings underscore the versatile, context-dependent antileukemic potential of PPARα signaling across diverse blood cancer subtypes.

Despite these findings, the potential of PPARα to regulate the transcriptional landscape of lymphoid leukemias—specifically through crosstalk with the NOTCH1 and STAT3 signaling networks—remains largely unexplored. This suggests that targeting the intersection of metabolism and transcription via PPARα could represent a novel vulnerability in T-ALL.

In this study, we propose a novel therapeutic strategy targeting a previously unrecognized PPARα-STAT3-NOTCH1 axis to overcome the limitations of current T-ALL treatments. Compared to traditional, experimental, or single-isoform PPARα agonists, chiglitazar—a novel PPAR pan-agonist with an exceptional safety profile currently approved by the NMPA for diabetes management-exhibits unique configuration-restricted binding properties that potentially expand its interactome beyond classical metabolic regulation. Mechanistically, we reveal that ligand-activated PPARα transcriptionally represses NOTCH1 not only by directly occupying its promoter region but also by physically interacting with STAT3 to impair its phosphorylation and subsequent nuclear translocation, thereby antagonizing STAT3-mediated NOTCH1 activation. This dual transcriptional blockade effectively circumvents the “on-target” gastrointestinal toxicities associated with direct GSI treatments. Moreover, given the frequent upregulation of Bcl-2 in T-ALL and its association with chemoresistance [20–22], we further demonstrate that chiglitazar induces a state of transcriptional priming, which significantly enhances the apoptotic sensitivity of T-ALL cells to venetoclax in both CDX and PDX models. Our findings establish PPARα as a critical negative regulator of the NOTCH1 signaling network and position chiglitazar as a highly promising, clinically translatable candidate for targeted combination therapy in aggressive T-ALL.

Methods

Reagents and cells

Chiglitazar was purchased from Chipscreen Biosciences Co., Ltd. (Shenzhen, China) and dissolved in dimethyl sulfoxide (DMSO) for use in all in vitro assays.

The Jurkat and Molt4 cell lines were obtained from the Key Laboratory of Hematologic Malignancies, Xiamen Municipal Key Laboratory of Hematologic Malignancies Diagnosis and Treatment, Department of Hematology, The First Affiliated Hospital of Xiamen University. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 U/mL penicillin (HyClone, Thermo Scientific, Massachusetts, USA) at 37 °C with 5% CO₂.

Cell viability and proliferation assays

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) (#HY-K0301, MCE). Briefly, 1 × 10⁴ Jurkat or Molt4 cells seeded in 96-well plates were treated with indicated concentrations of chiglitazar for 24–72 h, followed by incubation with CCK-8 reagent and absorbance measurement. Cell proliferation was quantified by Trypan Blue ( #72-57-1, Solarbio Technology Co., Ltd.) exclusion assay counting viable cells at specified time points.

Colony formation assay

Briefly, Jurkat or Molt4 cells were suspended in RPMI-1640 medium containing 0.35% low-melting agarose and seeded over a 0.5% agarose base layer in 12-well plates. After 14 days of culture, colonies were fixed with methanol, stained with 0.5% crystal violet (Yeasen Biotechnology Co., Ltd.), and counted using ImageJ.

Flow cytometric analysis of apoptosis and cell cycle

For apoptosis analysis, cells were stained with Annexin V-FITC and Propidium Iodide (PI) (#556547, BD Pharmingen) according to the manufacturer’s instructions. For cell cycle analysis, cells were fixed in 70% ethanol overnight, stained with PI/RNase solution, and analyzed using a flow cytometer to determine the distribution of cells in G0/G1, S, and G2/M phases.

RNA isolation and qPCR

Jurkat or Molt4 cells were seeded at a density of 2 × 10⁵ cells in 2 mL of culture medium in a 12-well plate and treated with DMSO or chiglitazar for 24 hours. Total RNA was extracted and purified using TRIzol reagent (Abclonal). Subsequently, 1 µg of RNA was reverse transcribed into cDNA using the ABScript III RT Master Mix for qPCR with gDNA Remover (Abclonal). qPCR was performed using 2X Universal SYBR Green Fast qPCR Mix (Abclonal). Analysis was carried out using the 2^−ΔΔCT method with GAPDH as the internal reference. The primers used for quantitative qPCR are as follows: GAPDH, forward 5’- GCTC ATTT GCAG GGGG GAG − 3’, reverse 5’- GTTG GTGG TGCA GGAG GCA − 3’; NOTCH1, forward 5’- GGAC CTCA TCAA CTCA CA -3’,reverse 5’- TTCT TCAG GAGC ACAA CT -3’; Hes1, forward 5’- AAGT CATC AAAG CCTA TTAT GG -3’, and reverse 5’- CTAT CTTT CTTC AGAG CATC C -3’; c-Myc, forward 5’- GCTG CTTA GACG CTGG ATTT − 3’, and reverse 5’- CACC GAGT CGTA GTCG AGGT − 3’.

Western blotting

Jurkat or Molt4 cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies against PPARα, NOTCH1, STAT3, and other targets, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence (ECL) system. The following primary antibodies were used: PPARα (Abcam); PPARγ (ABclonal); GAPDH (HUABIO); NOTCH1 (Abcam); NICD (Cell Signaling Technology); RPBJK (Abcam); Hes1 (Cell Signaling Technology); Hey1 (ABclonal); Bcl-2 (Cell Signaling Technology); Bcl-XL (Cell Signaling Technology); C-Myc (Cell Signaling Technology); STAT3 (Cell Signaling Technology); p-STAT3 (Cell Signaling Technology); Lamin B1 (Cell Signaling Technology); β-Tubulin (Proteintech).

RNA sequencing analysis

In brief, 1 × 10⁶ Jurkat cells were seeded in 10-cm dishes and treated with 30 µM chiglitazar for 24 h before being harvested for RNA sequencing by HaploX Biotechnology Co., Ltd.

DNA affinity precipitation assays

In brief, nuclear extracts were obtained in Jurkat or Molt4 cells treated with 20 µM chiglitazar using the NE-PER Kit (Thermo Scientific, #78835). 100 µg nuclear extracts combine with 2 µg of annealed biotinylated or unlabeled oligonucleotide (biotin-5′-ATGG AGGC CCAA GGTG GA-3′), and incubate overnight at 4 °C with 15 µl of Dynabeads M-280 magnet beads. Wash the beads and collect the bound proteins for Western blotting and mass spectrometry analysis.

Co-Immunoprecipitation (Co-IP)

Cell lysates were incubated with specific antibodies (anti- PPARα, anti-STAT3, or IgG control) and Protein A/G agarose beads overnight at 4 °C. The immunocomplexes were washed, eluted in SDS loading buffer, and analyzed by Western blotting to detect protein-protein interactions .

Chromatin Immunoprecipitation (ChIP)

ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit. Chromatin was cross-linked, fragmented, and immunoprecipitated with antibodies against PPARα or STAT3. The enrichment of NOTCH1 promoter regions was quantified by qPCR using specific primers (forward 5’-GAAT GGGC CACT CTC-3’ and reverse 5’-GTCT GCCC CAGC TGTG − 3’).

Luciferase reporter assay

HEK293T or leukemic cells were co-transfected with luciferase reporter plasmids containing the NOTCH1 promoter (or binding site mutants), along with PPARα or STAT3 expression vectors. Renilla luciferase vector was used for normalization. Luciferase activity was measured 24–48 h post-transfection using the Dual-Luciferase Reporter Assay System .

Animal studies

Six-week-old female NOD-Prkdc^−/−IL2rg^−/− (NOD/SCID) mice were obtained from the Laboratory Animal Center of Xiamen University. All mice were housed in a specific pathogen-free (SPF) facility, and experimental procedures were approved by the Laboratory Animal Ethics and Management Committee of Xiamen University.

To establish the CDX model, 6-week-old female NOD/SCID mice were subjected to 1 Gy of sublethal irradiation followed by intravenous (i.v.) injection of 3 × 10⁶ Jurkat cells. After cell inoculation, mice were randomly assigned to two groups (n = 7 per group). The treatment group received chiglitazar (15 mg/kg) via oral gavage for 12 consecutive days, while the control group received the vehicle. On day 12, three mice from each group were sacrificed to assess the infiltration of human CD45⁺ cells in the bone marrow, spleen, and peripheral blood using flow cytometry (anti-human CD45, clone HI30; anti-mouse CD45, clone 30F11; Biolegend). The remaining mice (n = 4 per group) were monitored for survival analysis.

For PDX establishment, NOD/SCID mice were inoculated with 1 × 10⁶ splenic cells harvested from humanized xenograft mice. Ten days post-transplantation, mice were randomized into two groups (n = 10 per group) and treated with either chiglitazar or vehicle by oral gavage for 15 consecutive days. Five mice from each cohort were sacrificed for flow cytometry, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) analyses, while the remaining animals were monitored for tumor progression. In a separate combination therapy experiment, the PDX model was established as described above. Ten days post-transplantation, mice were randomized into four groups (n = 5 per group) and treated for 14 days with vehicle, ABT199 (40 mg/kg/day), chiglitazar (10 mg/kg/day), or the combination. Treatment efficacy was evaluated via flow cytometry, HE staining, and IHC analyses. The clinical data of patients with T-ALL are summarized in Additional file 1: TableS1.

Statistical analysis

All data are presented as the mean ± standard deviation based on at least three independent experiments, each performed in triplicate. Comparisons between two experimental conditions were analyzed using the t-test, while two-way ANOVA was used for comparisons between more than two experimental conditions. All data analyses were performed using Prism 9 software. Statistical significance was considered when the p-value was less than 0.05.

Results

Chiglitazar activates PPARα and exhibits cytotoxic effects on T-ALL cells in vitro

Chiglitazar, a novel peroxisome proliferator-activated receptor pan-agonist, has been shown to have promising effects on glycemic control and lipid regulation in patients with type 2 diabetes [23]. Meanwhile, our previous study demonstrated that chiglitazar activates PPARα and exhibits anti-leukemia potential in AML [17].

To assess the biofunctions of chiglitazar in T-ALL cells, a Western blotting assay was first performed and we found that chiglitazar significantly enhanced the protein expression of PPARα in T-ALL cells (Jurkat and Molt4 cell lines), but not PPARγ (Fig. 1A). Next, we performed the CCK-8 assay (Fig. 1B) and trypan blue staining (Fig. 1C) to examine the cytotoxic effects of chiglitazar on Jurkat and Molt4 cells. Chiglitazar significantly inhibited cell proliferation and decreased cell viability in a dose- and time-dependent manner (Fig. 1B and C). Similarly, the EdU (5-ethynyl-2’-deoxyuridine) incorporation assay demonstrated that the cell proliferation was significantly decreased in chiglitazar-treated Jurkat and Molt4 cells (Fig. 1D). To confirm the functional requirement of PPARα in chiglitazar-mediated growth inhibition, we established PPARα-knockdown Jurkat and Molt4 cell lines. Subsequent CCK-8 assays revealed that the knockdown of PPARα significantly blunted chiglitazar-induced cytotoxicity and rescued cell viability in both T-ALL cell lines (Additional file 1: Fig. S1). These findings collectively demonstrate that the antileukemic activity of chiglitazar in T-ALL cells is predominantly mediated through PPARα. Taken together, these results suggest that the in vitro cytotoxicity of chiglitazar against T-ALL cells might be related to the activation of PPARα protein.

Fig. 1.

Chiglitazar activates PPARα and exerts cytotoxic effects on T-ALL cell lines in vitro. (A) Western blot analysis showing that chiglitazar treatment dose-dependently enhances PPARα protein expression in Jurkat and Molt4 cells, without significantly affecting PPARγ levels. (B-C) CCK-8 assay (B) and trypan blue staining (C) showing the viability and proliferation of Jurkat and Molt4 cells treated with increasing concentrations of chiglitazar at 24, 48, and 72 h, indicating dose- and time-dependent inhibition. (D) EdU incorporation assays confirming significantly reduced cell proliferation in Jurkat and Molt4 cells following 24 h of chiglitazar treatment. (E) Flow cytometry analysis of the cell cycle revealing that chiglitazar treatment for 24 h induces G0/G1 phase arrest in T-ALL cells. (F) Colony formation assays showing a significant reduction in the colony-forming ability of Jurkat and Molt4 cells treated with indicated concentrations of chiglitazar and cultured for 14 days. (G-H) Flow cytometry analysis using Annexin V/PI staining demonstrates that chiglitazar treatment for 24 h induces apoptosis in Jurkat and Molt4 cells in a dose-dependent manner. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Chiglitazar induces cell cycle arrest and increases apoptosis in T-ALL cells

To further investigate the potential mechanism underlying the cytotoxic effects of chiglitazar on T-ALL cells, flow cytometry analysis was performed to measure cell cycle. As shown in Fig. 1E and Additional file 1: Fig. S2, treatment with chiglitazar induced cell cycle arrest in G1 phase in Jurkat and Molt4 cells. Colony formation assays demonstrated that the number of colonies in Jurkat and Molt4 cells treated with chiglitazar was significantly reduced (Fig. 1F). Moreover, exposure to chiglitazar significantly increased apoptosis rates in Jurkat and Molt4 cells in a dose-dependent manner (Fig. 1G-H). Taken together, these results suggest that the in vitro cytotoxicity of chiglitazar against T-ALL cells might be associated with cell cycle arrest and increased apoptosis.

Chiglitazar inhibits the progression of T-ALL in vivo

To determine whether chiglitazar can inhibit the progression of T-ALL in vivo, we established a T-ALL CDX mouse model by intravenously injecting Jurkat cells into NOD/SCID mice (Fig. 2A). Compared to the control mice, chiglitazar treatment resulted in a significant reduction in the weight of the spleen (Fig. 2B-C). We further examined the infiltration of hCD45⁺ cells in the bone marrow (BM) and peripheral blood (PB) by flow cytometry. Flow cytometry analysis demonstrated that chiglitazar treatment significantly reduced the proportion of hCD45⁺ cells in both BM (Fig. 2D) and PB (Fig. 2E). Meanwhile, compared with vehicle-treated control mice, chiglitazar significantly improved the survival of the CDX mice (Fig. 2F). Continuous body weight monitoring in CDX mice demonstrated that chiglitazar did not cause pathological weight loss (Fig. 2G), and no other significant adverse reactions were observed, indicating favorable tolerability in mice. Taken together, these findings suggest that chiglitazar significantly delays leukemogenesis of T-ALL in vivo.

Fig. 2.

Chiglitazar inhibits T-ALL progression in a cell-line-derived xenograft (CDX) mouse model. (A) Schematic diagram of the experimental design for the Jurkat CDX mouse model. NOD/SCID mice were injected with Jurkat cells and treated with chiglitazar (15 mg/kg) or vehicle. (B-C) Representative images of spleens (B) and statistical analysis of spleen weights (C) showing significant reduction in splenomegaly in the chiglitazar-treated group compared to controls. (D-E) Flow cytometry analysis of human CD45⁺ (hCD45⁺) cell infiltration in the bone marrow (BM) (D) and peripheral blood (PB) (E), indicating reduced leukemic burden in treated mice. (F) Kaplan-Meier survival curves showing that chiglitazar treatment significantly prolongs the survival of CDX mice compared to the vehicle group. (G) Body weight monitoring of mice during treatment suggesting favorable tolerability with no significant pathological weight loss. (*p < 0.05, **p < 0.01)

Chiglitazar inhibits T-ALL progression by blocking the NOTCH1 signaling pathway

Next, to investigate the molecular mechanism by which chiglitazar inhibits T-ALL cells, RNA sequencing analysis was performed on Jurkat cells treated with DMSO or chiglitazar, respectively. Heatmap and GSEA (gene set enrichment) analysis results indicated that gene sets in Jurkat cells treated with chiglitazar were enriched in the NOTCH1 and HES pathway (Fig. 3A-C). The transcriptome profiling data from T-ALL and WT samples (GSE26530) [24] showed that the expression of NOTCH1 is significantly upregulated in T-ALL samples (Fig. 3D). These results suggest that chiglitazar might block the NOTCH1 pathway in T-ALL cells.

Fig. 3.

Chiglitazar suppresses the NOTCH1 signaling pathway, and NOTCH1 activation rescues Chiglitazar-induced cytotoxicity. (A-C) RNA sequencing and GSEA analysis of Jurkat cells treated with chiglitazar versus DMSO. Heatmaps (A) and enrichment plots (B-C) show significant enrichment and downregulation of NOTCH1 and HES pathway gene sets upon treatment. (D) Comparison of NOTCH1 mRNA expression between T-ALL patient samples and wild-type (WT) controls using the GSE26530 dataset. (E-H) qRT-PCR (E-G) and Western blot (H) analyses demonstrating that chiglitazar downregulates NOTCH1 and its downstream targets (Hes1, Hey1, c-Myc, Bcl-2, Bcl-XL) in Jurkat and Molt4 cells. (I-L) Overexpression of PPARα in Jurkat cells for 24 h mimics the effects of chiglitazar, suppressing NOTCH1 pathway proteins (I), inducing apoptosis (J-K), and inhibiting proliferation (L). (M-N) Rescue experiments showing that restoring NOTCH1 expression significantly attenuates the inhibitory effect of 24 h of chiglitazar on cell proliferation (M) and reverses the downregulation of c-Myc, Hes1, and Bcl-2 proteins (N). (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

To confirm this, we detected the expression levels of NOTCH1 and its target genes by western blotting and qRT-PCR, and the results showed that chiglitazar significantly inhibited the expression of NOTCH1 and its target genes such as RPBJK, Hes1, Hey1, c-Myc, Bcl2, and Bcl-XL in Jurkat and Molt4 cells (Fig. 3E-H). Meanwhile, overexpression of PPARα significantly suppressed the expression levels of NOTCH1, Hes1, Hey1, c-Myc, and BCL2 in Jurkat cells (Fig. 3I), further inducing apoptosis and inhibiting cell proliferation (Fig. 3J-L).

To evaluate the role of NOTCH1 in the chiglitazar-mediated inhibitory effect on T-ALL cell proliferation, the Jurkat cells were treated with chiglitazar and rescued expression of NOTCH1. The results showed that rescue expression of NOTCH1 significantly attenuated the chiglitazar-induced inhibitory effect on cell proliferation in Jurkat cells (Fig. 3M), and the suppression of c-Myc, Hes1, and Bcl-2 protein expression (Fig. 3N). These findings suggest that the cytotoxic effects of chiglitazar on T-ALL cell lines may be mediated, at least in part, through modulation of the NOTCH1 signaling pathway.

PPARα directly binds to the promoter region of the NOTCH1 gene and inhibits its transcriptional activity

To further explore the detailed molecular mechanisms by which PPARα inhibits the NOTCH1 pathway in T-ALL cells, we investigated its role as a transcription factor. PPARα regulates the transcription of various signaling pathways in multiple blood cancers [17, 18]. To examine the inhibitory effect of PPARα on NOTCH1 gene transcription, we measured the activity of a NOTCH1 promoter-driven luciferase reporter in 293T cells co-transfected with the reporter and a PPARα expression plasmid. As demonstrated in Fig. 4A, PPARα mediated a significant repression of the NOTCH1 gene promoter activity. Next, the binding sites (BS1 and BS2) of PPARα on the NOTCH1 gene promoter region were constructed based on the JASPAR database (Fig. 4B). Concurrently, the deletion of these binding sites significantly diminished PPARα-mediated repression of the NOTCH1 gene promoter (Fig. 4C), indicating that PPARα bound to both BS1 and BS2 sites to repress the NOTCH1 promoter. To further confirm the direct regulatory function of PPARα on the NOTCH1 promoter in T-ALL cells, we performed immunoprecipitation of chiglitazar-treated T-ALL cells using biotin-labeled oligonucleotide specific to BS1 (Fig. 4D), subsequently, mass spectrometry and western blotting analysis data indicate that PPARα could bind to BS1 and BS2 in Jurkat and Molt4 cells (Fig. 4E-F). Moreover, ChIP analysis confirmed that chiglitazar markedly increased the enrichment of PPARα on the NOTCH1 gene promoter (Fig. 4G). These results support our hypothesis that PPARα directly binds to the NOTCH1 promoter region and inhibits its transcriptional activity.

Fig. 4.

PPARα transcriptionally represses NOTCH1 by directly binding to its promoter and interacting with STAT3. (A) Luciferase reporter assay in HEK293T cells showing that PPARα overexpression significantly represses NOTCH1 promoter activity. (B-C) Identification of two PPARα binding sites (BS1 and BS2) in the NOTCH1 promoter (B). Deletion of these sites abolishes the repressive effect of PPARα on promoter activity (C). (D-F) DNA pull-down assay showing the experimental workflow using biotin-labeled oligonucleotides (D), liquid chromatography-mass spectrometry (LC-MS/MS) peak scores of PPARα-interacting complex proteins (E), and Western blot analysis confirming PPARα binding to BS1 and BS2 sites in Jurkat and Molt4 cells (F). (G) ChIP-qPCR analysis confirming increased enrichment of PPARα on the NOTCH1 promoter following chiglitazar treatment for 24 h. (H) Co-immunoprecipitation (Co-IP) assay demonstrating the physical interaction between PPARα and STAT3 in T-ALL cells. (I-L) Functional interplay between PPARα and STAT3. STAT3 overexpression for 24 h enhances NOTCH1 promoter activity (I), while PPARα overexpression (J) or chiglitazar treatment (K) antagonizes this STAT3-mediated activation. ChIP assay shows chiglitazar reduces STAT3 recruitment to the NOTCH1 promoter (L). (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

PPARα interacts with STAT3 and inhibits STAT3-mediated transcriptional activation of the NOTCH1 promoter

Multiple studies indicate that PPARα, in addition to directly regulating downstream genes as a transcription factor, can also modulate downstream genes by interacting with other transcription factors, such as HIF1α and p53 [17, 25]. The development of T-ALL is critically involved in the dysregulation of the JAK-STAT signaling pathway [26]. The results of mass spectrometry and Co-IP assays indicate that PPARα interacts with STAT3 in Jurkat and Molt4 cells (Fig. 4H).

Luciferase reporter assays demonstrated enhanced NOTCH1 promoter activity upon STAT3 activation, indicating that STAT3 functions as a transcriptional activator of the NOTCH1 gene (Fig. 4I). To test whether PPARα interacts with STAT3 and attenuates the transcriptional activity of NOTCH1 promoter, 293T cells were transfected with the expression plasmids of PPARα, STAT3, NOTCH1 promoter, Renilla luciferase plasmid, and a luciferase reporter assay was performed. As shown in Fig. 4J, the overexpression of STAT3 significantly enhanced the transcriptional activity of the NOTCH1 gene promoter, but this enhancement was suppressed by PPARα overexpression. Similarly, chiglitazar significantly attenuated STAT3-mediated transcriptional activation of the NOTCH1 gene in Jurkat cells (Fig. 4K). Moreover, ChIP data showed that chiglitazar markedly inhibited the recruitment of STAT3 on the NOTCH1 gene promoter (Fig. 4L). To further elucidate the precise molecular mechanism underlying PPARα-mediated inhibition of STAT3, we performed subcellular nuclear-cytoplasmic fractionation assays. As illustrated in Additional file 1: Fig. S3, chiglitazar treatment significantly suppressed the accumulation of phosphorylated STAT3 (p-STAT3) within the nuclear fraction of both Jurkat and Molt4 cells. These data suggest that the physical interaction between PPARα and STAT3 is likely responsible for restricting STAT3 phosphorylation and its subsequent nuclear activation. Collectively, these results demonstrate that STAT3 acts as a transcriptional activator of the NOTCH1 gene, whereas PPARα antagonizes this STAT3-mediated activation through interaction, thereby suppressing NOTCH1 transcription.

Chiglitazar inhibits T-ALL progression in a patient-derived xenograft mouse model

To confirm the effectiveness of chiglitazar against T-ALL, we established a T-ALL PDX using primary leukemia cells obtained from patients with T-ALL (Fig. 5A). PDX mice treated with chiglitazar exhibited a significant reduction in spleen weight compared with vehicle-treated controls (Fig. 5B-C). To evaluate the response to chiglitazar in this PDX model, we measured the percentage of hCD45⁺ cells by flow cytometry. Treatment with chiglitazar markedly reduced the levels of hCD45⁺ cells in the peripheral blood (PB), bone marrow (BM), and spleen (SP) (Fig. 5D-F). IHC (immunohistochemistry) and H&E (Hematoxylin-Eosin) staining confirmed that chiglitazar treatment substantially attenuated leukemic infiltration in both the spleen and liver (Fig. 5G-H, Additional file 1: Fig. S4A). To investigate the underlying mechanism, we assessed the protein levels of PPARα and NOTCH1 in spleen. IHC analysis demonstrated that chiglitazar treatment concomitantly upregulated PPARα and downregulated NOTCH1 (Fig. 5I-J, Additional file 1: Fig. S4A). Moreover, mice treated with chiglitazar exhibited improved survival rates compared to vehicle-controlled mice (Fig. 5K). Together, these results demonstrate that chiglitazar potently inhibits T-ALL disease progression in a patient-derived xenograft model.

Fig. 5.

Chiglitazar inhibits leukemia progression in a patient-derived xenograft (PDX) mouse model. (A) Schematic diagram of the T-ALL PDX mouse model established using primary leukemic cells from patients. (B-C) Assessment of tumor burden showing significantly reduced spleen size (B) and weight (C) in chiglitazar-treated PDX mice. (D-F) Flow cytometry quantification of hCD45 ⁺ cells reveals marked reduction of leukemic infiltration in peripheral blood (D), bone marrow (E), and spleen (F) following treatment. (G-H) Immunohistochemistry (IHC) for hCD45 (G) and H&E staining (H) confirm attenuated leukemic infiltration in the spleen and liver of treated mice. (I-J) IHC analysis of spleen sections showing upregulation of PPARα (I) and concomitant downregulation of NOTCH1 (J) in the chiglitazar-treated group. (K) Kaplan-Meier survival analysis demonstrating significantly improved survival in PDX mice treated with chiglitazar. (**p < 0.01, ***p < 0.001, ****p < 0.0001)

Synergistic suppression of T-ALL progression by chiglitazar and venetoclax in a PDX mouse model

To evaluate the clinical translational significance of chiglitazar in T-ALL treatment, we developed a combination therapy regimen of chiglitazar with venetoclax (Fig. 6A). Compared with the control group and the monotherapy groups, the combination therapy of chiglitazar and venetoclax significantly suppressed splenomegaly in the PDX mouse model (Fig. 6B-C). Flow cytometry analysis showed that the combination inhibited the infiltration of human CD45⁺ cells in the bone marrow (Fig. 6D-E) of PDX mice. Concomitant administration of chiglitazar and venetoclax synergistically reduced leukemic cell infiltration in both the spleen and liver of PDX mice (Fig. 6F-G, Additional file 1: Fig. S4B). Mechanistically, the combination of chiglitazar and venetoclax significantly increased the expression of PPARα and suppressed the expression of NOTCH1 in spleen of PDX mice (Fig. 6H, Additional file 1: Fig. S4B). These results suggest that the combination of chiglitazar and venetoclax has a beneficial therapeutic effect in the T-ALL PDX mouse and is at least partially attributable to PPARα/NOTCH1 pathway.

Fig. 6.

Chiglitazar synergizes with Venetoclax (ABT-199) to suppress T-ALL progression in vivo. (A) Schematic protocol for the combination therapy of chiglitazar and venetoclax in the PDX model. (B-C) Spleen images (B) and weight analysis (C) showing that the combination therapy suppresses splenomegaly more effectively than either monotherapy. (D-E) Representative flow cytometry plots (D) and quantification (E) of hCD45 ⁺ cells in the bone marrow, indicating a synergistic reduction in leukemic burden. (F-G) IHC analysis of hCD45 (F) and H&E staining (G) in spleen and liver tissues, confirming enhanced clearance of leukemic cells in the combination group. (H) IHC staining of spleen tissues demonstrating that the combination therapy leads to robust upregulation of PPARα and suppression of NOTCH1 expression. (ns, no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Discussion

Despite the incremental improvements in overall survival with intensified chemotherapy, T-ALL remains a clinical challenge, particularly due to the lack of effective targeted therapies for relapsed and refractory disease [1, 2]. The therapeutic landscape has long been dominated by the pursuit of NOTCH1 inhibitors; however, the clinical utility of direct enzymatic blockade (via γ-secretase inhibitors, GSIs) is severely compromised by prohibitive gastrointestinal toxicity and the rapid acquisition of resistance [9, 10].

In this study, we circumvent these historical barriers by identifying a non-canonical, transcription-dependent signaling mechanism—the PPARα-STAT3-NOTCH1 axis (Fig. 7). We demonstrate that the PPAR pan-agonist chiglitazar functions not merely as a ligand for nuclear receptor activation, but as a potent “transcriptional disruptor” that physically uncouples the STAT3-NOTCH1 positive feedback loop. This finding establishes a novel paradigm where nuclear receptors can be repurposed to target oncogenic transcription factors through direct protein-protein interference, independent of their classical metabolic roles [15, 27].

Fig. 7.

Schematic mechanism of Chiglitazar-induced NOTCH1 inhibition in T-ALL. Proposed model illustrating that chiglitazar activates PPARα, which directly binds to the NOTCH1 promoter and interacts with STAT3 to repress NOTCH1 transcription. This blockade of NOTCH1 signaling leads to the downregulation of downstream targets (Hes1, c-Myc, Bcl-2), resulting in cell cycle arrest, inhibition of proliferation, and induction of apoptosis in T-ALL cells

The constitutive activation of NOTCH1 is the master regulator of T-ALL pathogenesis, driving a transcriptional program that sustains leukemia stemness and proliferation [7, 28–30]. While GSIs attempt to block the proteolytic release of the intracellular domain (NICD), this approach fails to suppress the persistent transcriptional upregulation of the NOTCH1 gene itself, which is often reinforced by compensatory signaling from STAT3 [11, 29]. Our study provides novel mechanistic depth into how activated PPARα functions as a dominant-negative regulator of this circuit. Rather than a vague sequestration model, subcellular fractionation and Western blot analyses explicitly demonstrated that chiglitazar-induced PPARα activation effectively blocks the phosphorylation and subsequent nuclear translocation of STAT3. By restricting the nuclear accumulation of active p-STAT3 and simultaneously occupying the NOTCH1 promoter, PPARα dismantles the core transcriptional machinery required for NOTCH1 maintenance. This mechanism of “promoter occupancy and cofactor interference” represents a fundamental advance over GSIs. Unlike the indiscriminate inhibition of NOTCH receptors in healthy tissues, chiglitazar exerts a pathology-specific suppression of the aberrant NOTCH1 high-expression state found in leukemic blasts, thereby widening the therapeutic window [10].

Furthermore, our findings provide a mechanistic rationale for overcoming resistance to Bcl-2 inhibition, a growing concern in venetoclax-based therapies. Resistance to venetoclax in T-ALL is frequently driven by the compensatory upregulation of anti-apoptotic proteins like Mcl-1 and Bcl-XL [20–22]. We demonstrate that the chiglitazar-mediated blockade of the STAT3-NOTCH1 axis leads to the concomitant downregulation of these key anti-apoptotic guardians. By disrupting the upstream transcriptional network that sustains survival signaling, chiglitazar induces a state of enhanced apoptotic sensitivity rather than genetic synthetic lethality, effectively lowering the apoptotic threshold when combined with venetoclax [31], a synergy that conceptually parallels the co-targeting of Bcl-2 and PPARα in AML stem-like cells [18]. This aligns with recent evidence suggesting that targeting nuclear receptor signaling can sensitize hematologic malignancies to BH3 mimetics by remodeling the apoptotic machinery.

It is essential to contextualize these findings within the clinical translational framework and differentiate them from our group’s previous publications on chiglitazar in other blood cancers. While our previous studies demonstrated that chiglitazar exerts potent anti-leukemic effects in AML by disrupting the PPARα-HIF1α-PGK1 metabolic axis [17], enhancing apoptotic sensitivity through synergistic combination with Bcl-2 inhibitors [18], or triggering ferroptosis in leukemia stem cells when combined with HDAC inhibitors [19], the mechanism uncovered in aggressive T-ALL is entirely distinct and lineage-specific. T-ALL pathogenesis is uniquely dependent on the constitutive activation of the NOTCH1 signaling cascade —an oncogenic drive that is generally absent or non-essential in myeloid malignancies [29, 32]. Thus, the discovery of the PPARα-STAT3-NOTCH1 axis highlights a novel “non-canonical, transcription-disrupting” paradigm of chiglitazar, demonstrating that this nuclear receptor agonist can be leveraged to target distinct, context-dependent oncogenic vulnerabilities across different lineages of blood cancer. Furthermore, compared to traditional experimental PPARα agonists like fenofibrate or GW7647, chiglitazar’s existing NMPA approval for type 2 diabetes and its well-documented safety profile in patients drastically accelerate its clinical translational timeline, positioning it as an ideal partner for venetoclax in chemotherapy-free regimens for aggressive T-ALL.

In addition, the repurposing of chiglitazar highlights the potential of exploiting the “structural plasticity” of nuclear receptors for cancer therapy. While PPARα is traditionally viewed through the lens of lipid homeostasis, our data suggest its ligand-binding domain can be engaged to modulate diverse protein interactomes, including the JAK-STAT pathway. This concept is supported by recent literature demonstrating that nuclear receptors can function as “scaffolds” to repress inflammatory and oncogenic signal transducers [17], as exemplified by its synergy with either Bcl-2 inhibitors [18] or HDAC inhibitors [19] to disrupt stemness programs in AML. The ability of chiglitazar to inhibit T-ALL progression in PDX models without significant toxicity suggests that this non-canonical signaling function can be safely harnessed in the clinic.

However, there is currently insufficient evidence to determine whether PPARα can serve as a crucial molecule for prognostic evaluation and diagnostic subtyping in T-ALL. This urgently requires validation through larger, multicenter clinical cohorts to define its expression characteristics across different molecular subtypes and its correlation with clinical endpoints such as minimal residual disease (MRD) and relapse-free survival. Building upon this, the clinical benefit of therapeutically activating PPARα with chiglitazar is likely closely associated with the genetic background of the tumor, particularly in T-ALL patients harboring mutations in genes such as FLT3, DNMT3A, IDH1/2, or RUNX1 (mutations frequently observed in early T-cell precursor ALL or subtypes with myeloid/stem cell features) [4, 8, 32]. Therefore, it is necessary to systematically explore the synergistic or antagonistic relationships between these mutations and the PPARα pathway in preclinical models. Further verification of whether chiglitazar can confer additional anti-leukemic effects for specific mutant subpopulations is essential to facilitate the translation of PPARα from a potential biomarker into an actionable target for precision therapy.

Conclusions

In conclusion, our study identifies the PPARα-STAT3-NOTCH1 axis as a druggable vulnerability in T-ALL. We propose a therapeutic strategy that moves beyond enzymatic inhibition to transcriptional repression, offering a precise method to dismantle the core oncogenic circuitry of T-ALL. This approach not only provides a solution to the toxicity and resistance limitations of current NOTCH1 inhibitors but also positions chiglitazar as a promising partner for venetoclax in chemotherapy-free regimens.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AML

Acute myeloid leukemia

BM

Bone marrow

CCK-8

Cell Counting Kit-8

CDX

Cell line-derived xenograft

ChIP

Chromatin immunoprecipitation

Co-IP

Co-immunoprecipitation

CRi

Complete Remission with Incomplete Hematologic Recovery

DMSO

Dimethyl sulfoxide

ECL

Enhanced chemiluminescence

EdU

5-ethynyl-2’-deoxyuridine

FBS

Fetal bovine serum

GSIs

γ-secretase inhibitors

HE / H&E

Hematoxylin and eosin

IHC

Immunohistochemistry

NICD

NOTCH1 intracellular domain

NOD/SCID

NOD-Prkdc^−/−IL2rg^−/

PB

Peripheral blood

PDX

Patient-derived xenograft

PI

Propidium iodide

PPARα

Peroxisome proliferator-activated receptorα

SP

Spleen

STAT3

Signal transducer and activator of transcription 3

T-ALL

T-cell acute lymphoblastic leukemia

VDCLP

Vindesine/Daunorubicin/Cyclophosphamide/L-Asparaginase/Prednisone

WBC

White blood cell

WT

Wild-type

Author contributions

Jie Zha, Bing Xu, Wenjuan Li, Hui Zhou: study concept and design; Wenjuan Li, Hui Zhou, Dongmei Qin, Jiazhen Lin, Shuman Jia: experimental studies; Wenjuan Li, Hui Zhou, Dongmei Qin, Jie Zha, Bing Xu, Jianyu Weng: collection and data analysis and manuscript writing; Jie Zha, Bing Xu: obtained funding. All authors read and approved the final manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (U22A2090 and 82170180 granted for Bing Xu; 82470187 granted for Jie Zha) and the Natural Science Foundation of Fujian Province (2023J06054 granted for Jie Zha). Xiamen Municipal Bureau of Science and Technology (3502Z20234001 granted for Jie Zha; 3502Z20244015 granted for Bing Xu).

Data availability

The RNA-sequencing datasets generated and analyzed during this study are publicly available in the NCBI Gene Expression Omnibus (GEO) repository under the accession number GSE327005. The datasets presented in this study are available from the corresponding authors with reasonable requests.

Declarations

Ethics approval and consent to participate

All animal experiments have been supervised and approved by the Laboratory Animal Ethics and Management Committee of Xiamen University (XMULAC20220270).

Consent for publication

All authors have reviewed and approved the final version of the manuscript and consent to its submission to BMC Medicine.

Competing interests

The authors declare no competing interests.