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. 2025 Apr 4;25:129. doi: 10.1186/s12935-025-03757-3

Heme oxygenase 1 confers gilteritinib resistance in FLT3-ITD acute myeloid leukemia in a STAT6-dependent manner

Tianzhuo Zhang 1,3,#, Danna Wei 2,#, Yun Zhan 1,3,#, Zhengmei Long 1,3, Tingting Lu 4, Peng Zhao 1,3, Rui Gao 1,3, Qian Kang 1,3, Luxin Zhang 1,3, Min Liu 1,3, Xueying Yang 1,3, Jishi Wang 1,3,✉
PMCID: PMC11969713  PMID: 40186248

Abstract

Background

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. We previously discovered that heme oxygenase 1 (HO1) is crucial for chemoresistance in AML, but the detailed molecular mechanism of that remains unclear.

Methods

RNA sequencing was conducted to assess transcriptomic changes in three pairs of AML cells after regulating the expression of HO1. The molecular mechanism by which HO1 induces gilteritinib resistance in FLT3-ITD (FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)) AML was evaluated by quantitative real-time PCR (qRT-PCR), CCK-8, flow cytometry, and western blotting. FLT3-ITD AML mouse models were established to investigate the effects of HO1 expression on gilteritinib resistance in vivo.

Results

In these three pairs of AML cells, we discovered that HO1-mediated drug resistance is connected to the interleukin-4-mediated signaling pathway (specifically STAT6) only in MV4-11 cells with the FLT3-ITD mutation. Further findings revealed that HO1 overexpression confers gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens. While suppression of HO1 sensitized FLT3-ITD AML cell lines and primary individual specimens to gilteritinib. Mechanistically, western blotting and flow cytometry confirmed that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover, tumor-bearing mice were employed to determine that HO1 overexpression conferred gilteritinib resistance in vivo.

Conclusions

Collectively, these studies illustrate that HO1 may act as a successful treatment target for gilteritinib-resistant FLT3-ITD AML patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03757-3.

Keywords: Heme Oxygenase 1, Acute myeloid leukemia, FLT3-ITD mutation, Gilteritinib resistance, STAT6

Introduction

About 30% of adult acute myeloid leukemia (AML) patients comprise an extreme subgroup that is characterized by internal tandem duplications (ITDs) of the FLT3 receptor (FLT3-ITD). This mutation is linked to a worse prognosis and short survival rates [1–3]. Gilteritinib (ASP-2215), a tyrosine kinase suppressor of FLT3, ALK, and AXL [4, 5], has been authorized as a therapeutic strategy for FLT3-mutated AML cases [6], but drug resistance is still an issue. Multiple mechanisms, particularly FLT3 overexpression and/or the formation of new/secondary mutations, have been found to drive the progression of drug resistance to FLT3 inhibition [7]. We attempted to explain the processes behind gilteritinib resistance in FLT3-ITD AML and discovered the antioxidant stress factor heme oxygenase 1 (HO1) as a possible therapeutic target mediating resistance.

HO1, an enzyme that catalyzes heme breakdown, has a critical role in regulating oxidative stress, angiogenesis, apoptosis, chemoresistance, and proliferation in cancer cells [8–10]. We previously discovered that HO1 is crucial for chemoresistance in AML and that HO1 suppression increases the survival time of xenograft mice models [11, 12]. However, these findings have not been investigated in the context of gilteritinib resistance. Although changes in apoptosis and signaling pathway may play a role in drug response [11, 12], the precise contributions of HO1 in this process remain poorly known.

In this work, we discovered that HO1 confers gilteritinib resistance in FLT3-ITD AML in a STAT6-dependent way, therefore showing HO1’s hitherto unknown function as an antioxidant stress factor. We suggest that HO1 is a promising target for overcoming gilteritinib resistance in FLT3-ITD AML.

Materials and methods

Clinical specimens

Primary AML individual specimens were obtained from the Affiliated Hospital of Guizhou Medical University. Based on the Helsinki Declaration, informed permission was gained in every case. The Human Ethics Committee of the Affiliated Hospital of Guizhou Medical University authorized both the research and collection of specimens. PCR amplification and automated DNA profiling were employed to test all AML individual specimens for these gene mutations: FLT3-ITD, SF3B1, TCF, TP53, IDH1, IDH2, ZRSR2, RUNX1, GATA2, NPM1, SH2B3, TET2, DNMT3A, CEBPA, KMT2A, and C-kit. As previously stated, cytogenetics and identification of fusion genes by real-time PCR were also done [13, 14]. All six AML individuals with FLT3-ITD exclusively expressed the native FLT3-ITD and lacked detectable kinase domain alterations. Additional file 1: Table S1 summarizes the characteristics of the various AML individuals. Primary individual specimens were purified with Ficoll density centrifugation and were grown with the StemSpan Leukemic Cell Culture Kit (STEMCELL Technologies).

Cell cultures

Human cell lines K562 (FLT3 wild-type), THP-1 (FLT3 wild-type), MV4-11 (with FLT3-ITD mutation), and MOLM-13 (with FLT3-ITD mutation) were acquired from the Leibniz Institute DSMZ-German Collection of Micro-organisms and Cell Cultures (DSMZ). Both MV4-11 and MOLM-13 with FLT3-ITD exclusively expressed the native FLT3-ITD and lacked detectable domain alterations [15, 16]. The cell lines were assessed for contamination with mycoplasma and were confirmed by short tandem repeat profiling. Then, they were grown in RPMI-1640 medium mixed with 15% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. The incubation was conducted at 37 °C in a humidified atmosphere containing 5% carbon dioxide.

Reagents and antibodies

Gilteritinib (ASP-2215) and AS1517499 (a STAT6 suppressor) were acquired from Selleck Chemicals (USA). Gibco was the source of the RPMI 1640 medium and FBS (Carlsbad, CA, USA). Western blot was conducted using the anti-HO1 antibodies purchased from Abcam. The anti-cleaved PARP (C-PARP), anti-cleaved caspase3 (C-caspase3), anti-PARP, anti-caspase3, anti-p-STAT6, anti-STAT6, and anti-β-actin antibodies were obtained from Huabio (Hangzhou, China).

Quantitative real-time PCR (qRT-PCR)

This assay was carried out as stated before [17]. The qRT-PCR primers (Sangon Biotech, Shanghai, China) used in this study are summarized in Additional file 1: Table S2.

Western blot analysis

This assay was carried out as stated before [18].

Lentiviral transduction

Genechem Co., Ltd. supplied Lentivirus-hU6-MCS-Ubiquitin-firefly-Luciferase-IRES-puromycin (Shanghai, China). The HO1 overexpression lentivirus (LV-HO1) and HO1 silence lentivirus LV-HO1-RNAi (Si-HO1) were transduced into leukemia cells as per the manufacturer’s instruction. The controls were cells (primary AML cells, K562, THP-1, MV4-11, MOLM-13) transduced with the empty vector. Puromycin was employed to select stable cell lines expressing LV-HO1 or Si-HO1 after 5 days of maintenance and growth in RPMI-1640 media containing 15% FBS.

RNA sequencing (RNA-seq)

This assay was carried out as stated before [19].

Cell counting kit-8 (CCK-8) test

Cell viability was evaluated via the CCK-8 test. In short, 2 × 104 cells were treated with various drug concentrations for 24 h in a 96-well plate with a total volume of 100 µL. In the last 4 h of cell culture, 10 µL of CellTiter 96® AQueous One solution (Promega) was introduced to each well. At 490 nanometers, we evaluated the absorbance using a microplate spectrophotometer (BioTek). The drug-induced cell viability was represented as a percent relative to the control.

Apoptosis assay

This assay was carried out as stated before [20].

Xenografted tumor model

SPF (Beijing) Biotechnology Co., Ltd. provided male non-obese diabetic severely compromised immunodeficient (NOD/SCID) mice aged 6–8 weeks. Stably transduced MOLM-13luc cells were resuspended in PBS (5 × 106 cells/100 µL) and were subcutaneously administered to the mice randomized into 4 groups: EV4, LV-HO1, EV4 + gilteritinib, and LV-HO1 + gilteritinib. Mice were given gilteritinib (40 mg/kg/day, administered orally) every day for 27 days as soon as tumors were detectable or palpable [21]. The BLT In-Vivo Imaging System platform was used to observe the mice (BLT Photon Tech., Guangzhou, China). The tumor diameters and weights were determined every 3 days. The Institutional Animal Care and Use Committee of Guizhou Medical University authorized all researches on mice. This study avoided producing bias in the assessment of experimental results despite the absence of blinding.

Immunohistochemistry (IHC) staining

IHC staining using antibodies against Ki67 was conducted to determine protein levels. The quantification of protein expression level was conducted using ImageJ software.

Statistical analysis

Software GraphPad Prism 10.0 was employed for results analysis (GraphPad Software, Inc, USA). The normal distribution of the clinical data was determined by the Shapiro-Wilk normality test. Data are shown as mean ± SD for n = 3. A two-tailed, unpaired Student’s t-test was carried out to examine statistical differences between groups. The survival statistics were shown as Kaplan-Meier plots. A p-value < 0.05 revealed statistical significance.

Results

HO1-mediated drug resistance is related to the interleukin-4-mediated signaling pathway, especially to STAT6

We previously discovered that HO1 is crucial for chemoresistance in AML and that HO1 suppression increases the survival time of xenograft mice models [11, 12]. To comprehend the molecular mechanism of the role of HO1 on drug resistance in AML, further studies on the HO1 expression profile in leukemia cell lines were carried out. In comparison to other cell lines, the HO1 mRNA and protein expression levels were highest in MV4-11 cells, while its expression in K562 and THP-1 cells were lower (Fig. 1A, B, Additional file 1: Fig. S1A). HO1 expression was upregulated in K562 and THP-1 cells after transduction with LV-HO1, while HO1 expression was downregulated in MV4-11 cells after transduction with Si-HO1. Western blotting and qRT-PCR were utilized to assess the transduction ratio (Fig. 1C, D, Additional file 1: Fig. S1B).

Fig. 1.

Fig. 1

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HO1-mediated drug resistance is related to the interleukin-4-mediated signaling pathway, especially to STAT6. A, B In leukemia cells, HO1 protein and mRNA expression levels were tested. C, D HO1 was upregulated in K562 and THP-1 cell lines, but it was suppressed in MV4-11 cells. E Based on the RNA-seq data, the heatmap of hierarchical clustering displayed the differentially expressed genes between the MV4-11-EV and MV4-11-Si-HO1 groups. F GO analysis revealed a positive association between HO1 and gene signatures related to the interleukin-4-mediated signaling pathway. By the Student’s t-test, statistical differences were detected. ***p < 0.001

Using RNA-seq, we evaluated the transcriptome of the three pairs of transduced cell lines. The heatmap reveals the differently expressed genes between MV4-11-EV and MV4-11-Si-HO1 cells (Fig. 1E). Gene ontology (GO) enrichment analysis on the differentially expressed genes showed that HO1 is positively correlated with gene signatures involved with the interleukin-4-mediated signaling pathway, consisting of IL2RG, IL4R, JAK3, and STAT6 (Fig. 1F). It is well known that the interleukin-4-mediated signaling pathway is an important route in influencing drug resistance [22, 23]. We used the KEGG pathway database (https://www.genome.jp/kegg/pathway.html) to evaluate the role of IL2RG, IL4R, JAK3, and STAT6 in the interleukin-4-mediated signaling pathway. We noticed that STAT6 plays a vital role in this pathway, suggesting that HO1 overexpression may have a function in drug resistance in a STAT6-dependent manner.

HO1 upregulation enhanced the resistance of FLT3-ITD AML cell lines to gilteritinib

As mentioned above, we found that HO1 overexpression may play a part in drug resistance in a STAT6-dependent manner in MV4-11 cells exclusively. The MV4-11 cell line is a commonly used model for the FLT3-ITD mutant AML [24]. Thus, we hypothesized that HO1 overexpression contributes to treatment resistance in FLT3-ITD AML cell lines in a STAT6-dependent manner. The FLT3-ITD AML cells (MOLM-13, MV4-11) were employed to examine this assumption. The expression of HO1 mRNA and protein in the lentiviral-transduced MOLM-13 cells was assessed using qRT-PCR and western blotting. HO1 expression was elevated significantly in the LV-HO1 group when compared with the empty vector group, showing that the viruses were effectively transduced into MOLM-13 cells (Fig. 2A, Additional file 1: Fig. S2A). Apoptosis dysregulation is a key drug resistance mechanism. To study the anti-apoptosis function of HO1 in FLT3-ITD AML cells, we first measured cell viability following gilteritinib therapy in each group of MOLM-13 and MV4-11 cells using the CCK-8 test. Cell viability improved greatly in the LV-HO1 group, but it was substantially reduced in the Si-HO1 group (Fig. 2B). These findings indicate that the decreased expression of HO1 enhanced the growth-suppressive effects of gilteritinib on MV4-11 cells, and overexpression of HO1 diminished the inhibitory effects of gilteritinib on MOLM-13 cell growth.

Fig. 2.

Fig. 2

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HO1 upregulation enhanced the resistance of FLT3-ITD AML cell lines to gilteritinib. A In MOLM-13 cell lines, HO1 was overexpressed. B A CCK-8 test was employed to determine cell viability in the transduced MOLM-13 and MV4-11 cells after 24 h of gilteritinib administration. C Flow cytometry assay was employed to determine the apoptotic rate of the transduced MOLM-13 and MV4-11 cells after gilteritinib (10 nM) therapy for 24 h. D MOLM-13 and MV4-11 transduced cells were administered with gilteritinib (10 nM) for 24 h. Using western blotting, the expression of C-PARP and C-caspase3 was determined. By the Student’s t-test, statistical differences were identified. **p < 0.01, ***p < 0.001 and ##p < 0.01

Using flow cytometry, the apoptosis rate of MOLM-13 and MV4-11 cells was then determined. The findings indicate that HO1 overexpression can reduce gilteritinib-induced apoptosis in MOLM-13 cells, but suppressing HO1 can enhance the rate of gilteritinib-induced apoptosis in MV4-11 cells (Fig. 2C). These findings were further confirmed through western blotting (Fig. 2D, Additional file 1: Fig. S2B).

HO1 upregulation enhanced the resistance of FLT3-ITD primary AML individual specimens to gilteritinib

Ex vivo treatment was used on primary AML individual specimens that tested positive for FLT3-ITD to further support the clinical use of HO1 as a possible therapeutic target. From FLT3-ITD AML patients, primary AML cells were obtained and successfully transduced with either the control or Si-HO1/LV-HO1 lentiviruses. In line with FLT3-ITD AML cell lines, in the transduced primary cells, HO1 overexpression reduced the gilteritinib-induced apoptosis, while HO1 suppression promoted the gilteritinib-induced apoptosis (Fig. 3, Additional file 1: Fig. S3). These findings were further confirmed through western blotting (Fig. 4, Additional file 1: Fig. S4).

Fig. 3.

Fig. 3

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HO1 upregulation enhanced the resistance of FLT3-ITD primary AML individual specimens to gilteritinib. A Primary AML individual specimens (positive for FLT3-ITD, AML#1, #3, #5) were transduced with LV-HO1 or an empty vector. Flow cytometry was employed to evaluate the apoptotic rate of the transduced primary cells administered with gilteritinib (10 nM, 24 h). B Primary AML individual specimens (positive for FLT3-ITD, AML#2, #4, #6) were transduced with Si-HO1 or an empty vector. Flow cytometry was employed to assess the apoptotic rate of the transduced primary cells administered with gilteritinib (10 nM, 24 h). By the Student’s t-test, statistical differences were identified. **p < 0.01, ***p < 0.001, ##p < 0.01 and ###p < 0.001

Fig. 4.

Fig. 4

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HO1 upregulation enhanced the resistance of FLT3-ITD primary AML individual specimens to gilteritinib. A Western blotting was employed to assess the protein expression of C-PARP and C-caspase3 in transduced primary AML cells (AML#1, #3, #5) that had been handled with gilteritinib (10 nM) for 24 h. B Western blotting was employed to assess the protein expression of C-PARP and C-caspase3 in transduced primary AML cells (AML#2, #4, #6) that had been handled with gilteritinib (10 nM) for 24 h

HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens

To further explore the underlying molecular mechanism for the HO1-mediated gilteritinib resistance, we introduced the STAT6 suppressor (AS1517499) based on our.

previous findings. The findings illustrated that the usage of AS1517499 increased the apoptosis of MOLM-13-LV-HO1 cells as induced by gilteritinib, to some extent (Fig. 5A). This observation was further validated by elevated levels of C-PARP and C-caspase3 (Fig. 5B). Meanwhile, HO1 overexpression markedly increased the phosphorylation levels of STAT6 (p-STAT6) under gilteritinib treatment. While the addition of AS1517499 decreased p-STAT6 levels in MOLM-13-LV-HO1 cells to some extent (Fig. 5B). Similarly, HO1 downregulation markedly decreased the p-STAT6 in MV4-11 cells (Fig. 5C). These findings in FLT3-ITD AML cell lines were also confirmed in the FLT3-ITD primary AML individual specimens (Fig. 6, Additional file 1: Fig. S5). Overall, these data indicate that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens.

Fig. 5.

Fig. 5

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HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines. A The impact of STAT6 suppressor AS1517499 (100 nM, 24 h) on cell apoptosis of transduced MOLM-13 cells induced by gilteritinib (10 nM, 24 h). B The impact of AS1517499 (100 nM, 24 h) on the protein expression of p-STAT6, C-PARP, and C-caspase3 in transduced MOLM-13 cells with gilteritinib treatment (10 nM, 24 h). The histogram displays the relative gray values. C Western blotting was employed to investigate the impact of HO1 inhibition on the phosphorylation levels of STAT6 in MV4-11 cells. The histogram displays the relative gray values. By the Student’s t-test, statistical differences were identified. **p < 0.01, ***p < 0.001, #p < 0.05 and ##p < 0.01

Fig. 6.

Fig. 6

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HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD primary AML individual specimens. A The impact of AS1517499 (100 nM, 24 h) on gilteritinib-induced apoptosis of the transduced primary AML cells (AML#1, #3, #5) (10 nM, 24 h). B The impact of AS1517499 (100 nM, 24 h) on the protein expression of p-STAT6, C-PARP, and C-caspase3 in transduced primary AML cells (AML#1, #3, #5) with gilteritinib treatment (10 nM, 24 h). C Western blotting was employed to evaluate the impact of HO1 inhibition on the phosphorylation levels of STAT6 in primary AML cells (AML#2, #4, #6). By the Student’s t-test, statistical differences were identified. **p < 0.01, ***p < 0.001, #p < 0.05 and ##p < 0.01

HO1 upregulation enhanced the resistance of FLT3-ITD AML to gilteritinib in vivo

To investigate the role of HO1 in gilteritinib resistance in vivo, LV-HO1 or empty vector-transduced MOLM-13 cells were subcutaneously administered to mice as xenograft models. Gilteritinib was given orally to the mice immediately after being able to palpate the tumor (Fig. 7A). The findings demonstrated that, in comparison to the EV4 group, tumor proliferation was increased by HO1 overexpression (Fig. 7B). Moreover, HO1 overexpression significantly increased the tumor volume (Fig. 7C), tumor weight (Fig. 7D), and cell proliferation (Ki67+, Fig. 7F). Interestingly, gilteritinib therapy resulted in a substantial reduction in tumor load. Furthermore, mice in the EV4 + gilteritinib group exhibited longer survival, while mice in the other three groups had a similar and shorter survival time (Fig. 7E). In conclusion, our results demonstrate that upregulation of HO1 expression elevates the resistance of FLT3-ITD AML to gilteritinib in vivo.

Fig. 7.

Fig. 7

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HO1 upregulation enhanced the resistance of FLT3-ITD AML to gilteritinib in vivo. A MOLM-13luc mice trials are illustrated in a schematic. B Representative MOLM-13luc mice pictures in each group. C Growth curves of tumor volume for subcutaneous xenografts. D Tumor weight of MOLM-13luc mice in each group. E Survival curves of MOLM-13luc mice in each group. F Xenograft tumors in each group were stained with HE and Ki67 (HE, ×20 magnification; Ki67, ×20, ×40 magnification). The histogram displays the Ki67 IHC scores. By the Student’s t-test, statistical differences were identified. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05 and ##p < 0.01

Discussion

This investigation revealed the biological functions of HO1 in FLT3-ITD AML. HO1 overexpression provides gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens, according to the data. And suppression of HO1 caused FLT3-ITD AML cell lines and primary individual specimens to be more susceptible to the cytotoxic effects of gilteritinib. Western blotting and flow cytometry verified that the HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover, mice with tumors were employed to confirm that HO1 overexpression provides gilteritinib resistance in vivo. These findings reveal that HO1 could be an appropriate treatment target for FLT3-ITD AML patients who are resistant to gilteritinib (Fig. 8).

Fig. 8.

Fig. 8

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Schematic representation of HO1-mediated resistance of FLT3-ITD AML to gilteritinib. HO1-mediated gilteritinib resistance is involved with STAT6 phosphorylation in FLT3-ITD AML. Administration of AS1517499 could overcome the gilteritinib resistance to some extent

Regrettably, due to the limitations of laboratory conditions and lack of appropriate experimental tools, 32D or BaF3 cell lines stably expressing human FLT3 with different mutations were not used in our research. During mutagenesis using 32D or BaF3 cells, stress protein p38 and changes in HO1 activity should be identified, which is significant for exploring the relationship between FLT3 mutations and the oxidative stress pathway. We hope to conduct related experiments in the future when experimental conditions improve.

As previously discussed [25], HO1 is an enzyme that degrades dying cells to liberate heme and create CO, Fe2+, and biliverdin as a result. Moreover, HO1 is essential for avoiding cellular injury. The antioxidant, anti-apoptotic, and anti-inflammatory capabilities of its metabolites promote cell adaptability to various stresses. It inhibits apoptosis during chemotherapy of malignant tumors and promotes tumor development [26–30]. Earlier, we established that HO1 inhibits AML cell apoptosis through the JNK/c-JUN signaling pathway [11]. However, the link between the HO1 and STAT6 signaling pathway in FLT3-ITD AML has not been investigated in depth.

Until now, many investigations regarding the contribution of HO1 in the apoptotic signaling pathway in AML have concentrated on the relationship between HO1 and NF-κB or Nrf2 [31, 32]. In direct opposition, HO1 has rarely been related to the STAT6 pathway, which is involved in cell differentiation, apoptosis, and proliferation [33–35]. The STAT6 protein has a crucial function in the regulation of inflammatory responses related to carcinogenesis [36]. The STAT6 protein is triggered when Janus kinase (JAK) family proteins are phosphorylated in reaction to the attachment of interleukin (IL)-4 or IL-13 to their shared type II IL-4 receptor. Upon activation, STAT6 elicits a double role in signaling and transcription through the dimerization of phosphorylated STAT6 and subsequent translocation to the nucleus, which in turn activates several genes [37]. This signaling pathway is crucial for different cell kinds, including tumor cells [37, 38]. Furthermore, in refractory or relapsed diffuse large B-cell lymphoma, an elevated phospho-STAT6 protein has been associated with treatment resistance and has been an appealing target for therapeutic strategies [39]. To our knowledge, this is the study to show that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML in vitro and in vivo. Administration of AS1517499 could overcome the gilteritinib resistance to some extent. Indeed, the detailed molecular mechanisms of HO1 in regulating the phosphorylation of STAT6 warrants future studies.

FLT3 inhibitors (FLT3i) are classified into the first or second type according to the binding mode to FLT3. Type I inhibitors, which include midostaurin, gilteritinib, lestaurtinib, and crenolanib, bind the FLT3 receptor in the active conformation near the activation loop or the ATP binding domain. Type I inhibitors have the capacity of inhibiting both FLT3-ITD and FLT3-TKD. On the other hand, type II inhibitors, including quizartinib and sorafenib, bind the FLT3 receptor in the inactive conformation in a region adjacent to the ATP-binding domain. As these mutations modify the active kinase conformation of FLT3, type II inhibitors are inactive against most FLT3-TKD mutations [40]. Mechanisms involved in resistance could be heterogenous for different FLT3i and grouped as FLT3-dependent and FLT3-independent resistance mechanisms, which are not required to be equally or mutually present.

Previous studies reported that FLT3i resistance can be classified into FLT3-dependent and -independent mechanisms [41]. FLT3-dependent mechanisms include the modulation of FLT3-ITD localization or secondary domain mutations. For example, the presence or emergence of FLT3-TKD or FLT3-ITD-TKD (compound) mutations before or during FLT3i treatment causes the development of resistance against FLT3i therapy. The gatekeeper mutation, F691L, in the TKD is the common one that led to resistance against all clinically used FLT3is [42, 43]. Gilteritinib was shown not to be effective in patients carrying F691L mutation who enrolled in the ADMIRAL study [44]. Additional mutations in the FLT3 receptor account for a small proportion of resistant cases. Studies elucidating FLT3-independent resistance mechanisms, such as the impact of the tumor microenvironment, metabolism of FLT3i, and modulation of alternative intracellular signaling pathways, have been paid attention [45]. The purpose of this article is to elucidate the mechanism by which HO1 mediates resistance to gilteritinib in FLT3-ITD AML from a FLT3-independent perspective. Future studies to confirm the HO1 expression in primary leukemic cells necessitates a larger sample size of FLT3-ITD AML patients. Furthermore, it is essential to clarify the underlying signaling pathway, since the mechanism by which HO1 resists cell apoptosis through the STAT6 pathway remains obscure.

Conclusion

Taken together, our results imply that HO1 works as an oncoprotein in the progression of FLT3-ITD AML and may act as a potential curative biomarker in the future.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (2.6MB, docx)

Acknowledgements

We thank Bullet Edits (http://www.bulletedits.cn) for its linguistic assistance during the preparation of this manuscript.

Abbreviations

AML

Acute myeloid leukemia

HO1

Heme oxygenase 1

FLT3-ITD

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)

qRT-PCR

quantitative real-time PCR

RNA-seq

Ribonucleic acid sequencing

CCK-8

Cell counting kit-8

IHC

Immunohistochemistry

mRNA

messenger RNA

GO

Gene ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

CO

Carbon monoxide

Author contributions

JSW and TZZ were involved in the study conception and design. DNW, YZ, ZML, TTL, and PZ performed the experiments. RG, QK, LXZ, ML, and XYY interpreted the data. TZZ wrote the original manuscript. All authors revised the manuscript. All authors read and approved the final manuscript.

Funding

The present study was supported by the National Natural Science Foundation of China (No. 82460039, No. 82370168, and No. 82170168), Guizhou Provincial Basic Research Program (Natural Science) (No. QianKeHe-zk[2025] General-451), Science and Technology Program of Guizhou Province Health Committee (No. gzwkj2024-365 and No. gzwkj2025-090), and Doctoral Research Startup Fund of Affiliated Hospital of Guizhou Medical University (gyfybsky-2024-24).

Data availability

The data used and/or analysis during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The animal care and ethical committee of Guizhou Medical University approved our protocols. All animal-handling procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and followed the guidelines of the Animal Welfare Act. According to the Helsinki Declaration, the informed consent was first obtained in writing. All patients volunteered to participate in this experiment. All patient information has been kept confidential.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Tianzhuo Zhang, Danna Wei and Yun Zhan contributed equally to this work.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (2.6MB, docx)

Data Availability Statement

The data used and/or analysis during the current study are available from the corresponding author on reasonable request.

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