==== Front
Cell Death Discov
Cell Death Discov
Cell Death Discovery
2058-7716
Nature Publishing Group UK London

1528
10.1038/s41420-023-01528-5
Review Article
NADPH oxidase mediated oxidative stress signaling in FLT3-ITD acute myeloid leukemia
http://orcid.org/0000-0003-4488-0380
Chen Yongfeng cyfeng@tzc.edu.cn

1
http://orcid.org/0009-0000-5944-2901
Zou Zhenyou Zouzy@glmc.edu.cn

2
http://orcid.org/0000-0001-7133-8875
Găman Mihnea-Alexandru mihneagaman@yahoo.com

34
Xu Linglong 5
Li Jing 6
1 grid.440657.4 0000 0004 1762 5832 Department of Basic Medical Sciences, Medical College of Taizhou University, Taizhou, Zhejiang 318000 China
2 Institute of Psychosis Prevention, Brain Hospital of Guangxi Zhuang Autonomous Region, Liuzhou, Guangxi 542005 China
3 grid.8194.4 0000 0000 9828 7548 Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
4 grid.415180.9 0000 0004 0540 9980 Department of Hematology, Centre of Hematology and Bone Marrow Transplantation, Fundeni Clinical Institute, Bucharest, Romania
5 grid.452858.6 0000 0005 0368 2155 Department of Hematology, Taizhou Central Hospital (Taizhou University Hospital), Taizhou, Zhejiang 318000 China
6 grid.449525.b 0000 0004 1798 4472 Department of Histology and Embryology, North Sichuan Medical College, Nanchong, Sichuan 637000 China
30 6 2023
30 6 2023
2023
9 20818 3 2023
14 6 2023
22 6 2023
© The Author(s) 2023
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
The internal tandem duplication of the juxtamembrane domain of the FMS-like tyrosine kinase 3 (FLT3-ITD) is the most common genetic change in acute myeloid leukemia (AML), and about 30% of all AMLs harbor a FLT3-ITD mutation. Even though FLT3 inhibitors have displayed encouraging effects in FLT3-ITD-mutated AML, the extent of the clinical response to these compounds is cut short due to the rapid development of drug resistance. Evidence has shown that FLT3-ITD triggered activation of oxidative stress signaling may exert a pivotal role in drug resistance. The downstream pathways of FLT3-ITD, including STAT5, PI3K/AKT, and RAS/MAPK, are considered to be major oxidative stress signaling pathways. These downstream pathways can inhibit apoptosis and promote proliferation and survival by regulating apoptosis-related genes and promoting the generation of reactive oxygen species (ROS) through NADPH oxidase (NOX) or other mechanisms. Appropriate levels of ROS may promote proliferation, but high levels of ROS can lead to oxidative damage to the DNA and increase genomic instability. In addition, post-translational modifications of FLT3-ITD and changes in its subcellular localization can affect downstream signaling which may also be one of the mechanisms leading to drug resistance. In this review, we summarized the research progress on NOX mediated oxidative stress signaling and its relationship with drug resistance in FLT3-ITD AML, and discuss the possible new targets in FLT3-ITD signal blocking to reverse drug resistance in FLT3-ITD-mutated AML.

Subject terms

Oncogenesis
Mechanisms of disease
issue-copyright-statement© Cell Death Differentiation Association (ADMC) 2023
==== Body
pmcFacts

FLT3-ITD-triggered activation of oxidative stress signaling may play a critical role in drug resistance in AML.

Post-translational modification of FLT3-ITD and changes in its subcellular localization can affect downstream signaling which may be one of the mechanisms leading to drug resistance in AML.

Open questions

What is the exact mechanism underlying the alteration of downstream signaling of the FLT3-ITD oncogene?

What is the exact relationship between post-translational modifications, subcellular localization, and FLT3-ITD oncogene signaling?

How to efficiently and persistently block FLT3-ITD oncogene signaling?

Introduction

Abnormal signals from receptor tyrosine kinases (RTKs) are often discovered to exert a pivotal role in tumor initiation and progression. FLT3 protein belongs to the type III RTKs family and is solely expressed in normal bone marrow CD34+ stem/progenitor cells [1, 2]. The most frequent mutation that involves FLT3, which can occur in up to 30% of acute myeloid leukemia (AML) cases, is the internal tandem duplication (ITD) of its juxtamembrane domain [3]. The FLT3-ITD mutation causes constitutive activation of the tyrosine kinase domain, resulting in the autophosphorylation of the receptors and consequently in the phosphorylation of substrate proteins which in turn activate downstream pro-survival signaling pathways such as phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT), Ras/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT), promoting the progression of leukemia [4–6]. Currently, it is well accepted that FLT3-ITD is an oncogenic driver mutations and a marker of poor prognosis in AML. According to a recent risk stratification study for AML patients, individuals with FLT3-ITD were less likely to achieve complete remission (OR) and were at increased risk of relapse-related mortality [7]. Accumulating evidences suggest that FLT3-ITD signal induced oxidative stress may involve in the pathogenesis of FLT3-ITD AML [8–10].

Several publications have delineated that the FLT3-ITD mutation is closely linked with an increased ROS production in AML cells [11, 12]. As a second signaling molecule, the role of ROS in FLT3-ITD AML is manifold. Moderate levels of ROS can act as pro-survival signals to promote cell proliferation. However, high concentrations of ROS can be responsible for oxidative damage and increase the probability of an erroneous repair of the DNA [13]. It is worth noting that quiescent AML LSCs generally have low ROS levels compared with cycling LSCs and bulk AML cells [14]. However, quiescent AML LSCs still inevitably suffer from oxidative DNA damage even in the absence of high ROS levels. Such damage increases genomic instability and drives the genetic evolution of LSCs, which is likely to give them competitive advantages [15]. Evidence of cytogenetic evolution has shown that the rapid clonal evolution through genomic instability may be the main reason for the high relapse rate and chemorefractoriness in AML [16]. In a recent study, Schmalbrock et al. investigated the clonal evolution patterns of FLT3 ITD AML patients receiving Midostaurin treatment to explore the potential mechanisms for tyrosine kinase inhibitors (TKI) resistance. The results showed that more than half of the patients acquired new mutations at the time of disease resistance or progression. However, in 32% of cases, no FLT3-ITD mutational change was observed, suggesting there are other resistance mechanisms which can bypass FLT3 inhibition [6].

Recently, numerous studies have shown that the post-translational modifications that involve FLT3-ITD and its localization in cells can lead to changes in downstream signaling which may also be another mechanism leading to TKI drug resistance. Therefore, effective targeting of FLT3-ITD in the personalized approach to AML management necessitates an in-depth understanding of the underlying links between FLT3-ITD localization, activity, and downstream signaling.

FLT3-ITD subcellular localization

The FLT3 gene is located on chromosome 13q12 in humans. This gene encodes a protein consisting of 993 amino acids, whose structure is made up by an “N-terminal extracellular domain, a transmembrane region, a juxta-membrane domain, and a C-terminal cytoplasmic tyrosine kinase domain” [4]. The FLT3 protein is co-translationally translocated into the endoplasmic reticulum (ER) and is then subjected to a multistep glycosylation process and folding at the ER. The control system in the ER guarantees that solely correctly folded FLT3 molecules leave the ER, undergo further glycosylation in the Golgi apparatus, and ultimately move into the plasma membrane (PM) [17].

Schmidt-Arras et al. highlighted the existence of two forms of the FLT3 receptor via western blotting analysis. One 150 kDa receptor represents a complex glycosylated mature form, and the other 130 kDa receptor represents an underglycosylated immature form with a mannose-rich structure. The 130-kDa forms were predominantly detected in cells expressing FLT3-ITD, whereas 150 kDa forms were found to have a higher proportion in the cells expressing FLT3-WT [18]. Multiple studies have shown that activated mutations lead to altered subcellular localization of the FLT3 protein. Several investigations have demonstrated that the FLT3 wild-type protein is mainly located at the PM, while the FLT3-ITD protein is mainly detected in the ER, Golgi apparatus, and perinuclear region, but rarely or even unable to be detected in the PM [19, 20]. Evidence suggests that an altered localization of FLT3-ITD may affect its downstream signaling and may be associated with the development of drug resistance [20–22].

Currently, the mechanism by which activated mutations lead to altered FLT3-ITD subcellular localization is unclear. Besides being possibly related to the glycosylation status, recent studies have found that palmitoylation and phosphorylation may be two other key factors regulating FLT3-ITD intracellular localization. According to Lv et al., the S-palmitoylation of FLT3-ITD is responsible for the inhibition of FLT3-ITD’s surface expression, and the application of the palmitoylation inhibitor 2-bromopalmitate (2-BP) can decrease the ER retention of FLT3-ITD and promote its transportation to Golgi and cell surfaces [19]. Similarly, the application of tyrosine kinase inhibitors (TKIs) can also increase FLT3-ITD’s plasma membrane expression levels, which may be due to the fact that TKIs can inhibit the phospho-dependent interaction of FLT3-ITD with ER resident proteins and promote FLT3-ITD glycosylation [18, 19, 23]. Elevated FLT3-ITD plasma membrane expression levels following the use of TKIs have been reported to enhance the efficacy of chimeric antigen receptor T-cell therapy and antigen-dependent cytotoxicity based on anti-FLT3 compounds [23], suggesting that combining TKIs with immunotherapy may help improve prognosis for AML patients.

NOX play a key role in FLT3/ITD signaling

AML is a group of hematologic malignancies with high heterogeneity which is also extended to their metabolic profiles [24–26]. It was reported recently that FLT3/ITD induce a significant increase in aerobic glycolysis via AKT-mediated upregulation of mitochondrial hexokinase (HK2), and render AML cells highly dependent on glycolysis [25]. However, another study of Erdem et al. showed that compared to FLT3wt AML cells, which are reliant on glycolysis, FLT3-ITD AML cells are more OXPHOS-driven [26]. Besides, studies also showed that AML LSCs seem to mainly rely on mitochondrial respiration, therefore, may actually be particularly sensitive to OXPHOS inhibition [14, 26].

ROS are an important by-product of aerobic metabolism. Excessive production of ROS is frequently observed in FLT3-ITD AML and is demonstrated closely associated with the pro-survival mechanism of AML cells [27–29]. In eukaryotic cells, ROS can be produced by a number of approaches, including mitochondria, the xanthineoxidase system, and the NOX system, etc. [13]. However, it was reported that in AML cells, the increase of ROS is mainly attributable to the constitutive activation of NOX. In contrast, overproduction of mitochondrial ROS was rarely observed [27–29]. NOX is a multienzyme complex that uses cytoplasmic NADPH as electron donor to catalyze the production of the superoxide anion (O2−) from extracellular O2 [30]. NOX includes 7 isoforms, i.e., dual oxidase (DUOX) 1–2 and NOX1-5. Each isoform displays different mechanisms of regulation. The first four isoforms (NOX1-4) all require binding to p22Phox (a membrane bound NOX complex subunit) to secure an accurate post-translational modification, long-term stability and membrane targeting. Furthermore, with the exception of NOX4, all these proteins require additional regulatory subunits for their activity, which have to translocate from the cytosol to form a functional complex with the membrane-bound NOX [30, 31].

Among all NOX isoforms, NOX1, NOX2 and NOX4 are expressed by human CD34+ hematopoietic progenitor cells [30–32]. Among them, NOX2 and NOX4 are critical players in the generation of ROS via FLT3-ITD signaling [11, 33]. Sallmyr el al. found that FLT3-ITD signaling promotes binding of Rac1 GTPase to phosphorylated STAT5 (pSTAT5) in FLT3-ITD-transfected and positive cells, which in turn enhances NOX2 activity and promotes ROS production. Application of FLT3 inhibitors blocked ROS generation, resulting in reduced double-stranded DNA breaks (DSBs), and enhanced efficiency and fidelity of the DNA repair processes [8]. Likewise, there is evidence that FLT3-ITD signaling promotes NOX4 expression and the generation of ROS via STAT5 mediation. Furthermore, it facilitates the inactivation of one of its negative regulators, that is, the protein-tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1), by reversible oxidation of its catalytic cysteine residue. NOX4 knockdown reduces ROS concentrations, restores the activity of DEP-1, and reduces FLT3-ITD-driven transformation [33].

Recently, Moloney et al. reported that PM-located FLT3-ITD can promote NOX4 activation by activating AKT, thereby inducing the production of pro-survival ROS. This process depends on the AKT activation and only PM- located FLT3-ITD can activate AKT. Therefore, it is believed that only FLT3-ITD at the PM is carcinogenic, and targeting PM-localized FLT3-ITD may prove beneficial in the prevention of downstream ROS-driven carcinogenesis [11]. In another study, Lv et al. used 2-BP to inhibit the palmitoylation of FLT3-ITD and discovered that the enhanced expression of FLT3-ITD in the PM can promote oncogene signaling mediated by AKT and other pathways, thereby promoting the proliferation of leukemia cells [19]. The result of the study by Lv et al. confirmed that the PM-located FLT3-ITD is the main source of oncogene signaling. However, there is evidence indicating that oncogene signaling is originated not only from the PM-located FLT3-ITD, but also from the FLT3-ITD located in early secretory compartments [20]. Currently, the subcellular localization of FLT3-ITD and its impact on downstream signaling pathways has become a hot topic in AML research, an in-depth understanding of the FLT3-ITD signal and its downstream pathways will help to precisely suppress the transmission of AML oncogene signals.

Impact of subcellular localization of FLT3-ITD on downstream signaling pathway

The downstream signaling pathways of the FLT3-ITD oncogene, e.g., PI3K/AKT, JAK/STAT, and Ras/ERK, are representative signaling pathways that are abnormally activated in AML. The aberrant FLT3-ITD oncogene signal constitutively activates the above signaling pathways, which subsequently inhibit cell apoptosis, promote cell proliferation and survival via regulation of apoptosis-related genes, thereby leading to the initiation and development of AML [34–37].

Recently, several studies have highlighted that the intracellular disposition of FLT3-ITD may determine which downstream pro-survival signals are activated constitutively by FLT3-ITD. In particular, the PM-localized FLT3-ITD strongly activates the mitogen-activated protein kinase (MAPK) and PI3K pathways with diminished STAT5 phosphorylation [20]. Golgi-localized FLT3-ITD activates STAT5, AKT and ERK [19, 21], whereas ER-retained FLT3-ITD aberrantly activates STAT5 and upregulates its targets, Pim-1/2, but fails to activate AKT and ERK signaling [21, 22] (Fig. 1). Currently, it is believed that resistance to TKIs can also develop as an “off-target” mechanism when changes occur in the subcellular localization of FLT3-ITD and result in alterations of downstream pathways [20–22]. In the study of Fleischmann et al., pre-treatment with N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) led to intracellular retention of FLT3-ITD, and the susceptibility of FLT3-ITD-expressing AML cells to 17-AAG (HSP90 inhibitor) was increased. 17-AAG treatment induced degradation of FLT3-ITD and promoted apoptosis in AML cells. In contrast, histone deacetylase inhibitor valproic acid (VPA) pre-treatment significant increased the surface expression of FLT3-ITD, phosphorylated ERK and phosphorylated AKT were then increased, and the susceptibility of AML cells to rapamycin was enhanced [22]. The result of the study by Fleischmann et al. indicates that, the allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling might represent a new therapeutic strategy to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD AML.Fig. 1 NOX mediated oxidative stress signaling in FLT3-ITD AML.

The subcellular localization of FLT3-ITD may affect its downstream signaling (see main text, the dotted lines indicate a relatively weak effect) [20, 21]; The PI3K/AKT, STAT5, and ERK1/2 downstream pathways of FLT3-ITD can promote ROS generation through NOX4D localized at the nuclear membrane [11, 40]; FLT3-ITD signaling causes STAT5 phosphorylation, P-STAT5 subsequently translocates to the nucleus and activates the transcription of NOX4; P-STAT5 can also maintain the active form of RAC1, and promote RAC1 combination with NOX, leading to an increase in ROS generation; FLT3-ITD signaling also stabilizes p22phox protein via AKT pathway and increases ROS generation [93]. The dotted line in the figure indicates a relatively weak effect. This figure was drawn based on existing research data; its accuracy and more precise signaling mechanisms must be confirmed and supplemented by extensive, in-depth studies.

Ras/ERK, PI3K/AKT and JAK/STAT are the main oxidative stress signaling pathways and can induce pro-survival ROS production through different mechanisms, such as inhibiting the activity of forkhead O box (FOXO) or enhancing NOX expression [38, 39]. Recently, Moloney et al. found that in AML cells, the PI3K/AKT, STAT5, and ERK1/2 downstream pathways of FLT3-ITD can promote ROS generation through NOX4D (NOX4 28 kDa splice variant) localized at the nuclear membrane, and inhibiting these pathways could lead to a reduction in ROS production [40], suggesting that NOX4D localized at the nuclear membrane is the main source of pro-survival ROS production and is also a key factor leading to genomic instability and DNA damage [40].

Mounting evidence have shown that, secondary mutations caused by accumulation of DNA damage may critically contribute to the development of drug resistance [41–45]. However, the precise role of secondary mutations in relapse and TKIs resistance still requires further study. Recent several studies have showed that FLT3-ITD-induced ROS generation can result in the oxidation of tumor suppressor genes and of DEP-1 phosphatase which acts as negative regulator of FLT3. Moreover, mutagenesis can directly derive from DNA oxidation if DEP-1 inactivation is not repaired correctly [33, 46, 47]. Genomic instability mediated by ROS in FLT3-ITD AML progression has been excellently reviewed by Rebechi et al. [16].

Redox control is crucial to the survival of AML

ROS represent a group of oxygen-containing and highly reactive molecules which serve as important second messengers involved in a variety of signal transduction that regulates cell growth, proliferation, and differentiation of cells [13]. It is now accepted that the appropriate concentration of ROS is crucial for cancer cell survival, however, excessive ROS concentrations may cause both severe oxidative damage and eventually cell death [13]. Therefore, the maintenance of redox balance is crucial for any cell [48, 49].

Like other cell types, leukemia cells also have well-established and complex mechanisms for maintaining their redox balance, and the level of pro-survival ROS is finely regulated in AML. It was reported that in FLT3-ITD AML, elevated ROS concentrations induced by oncogene signaling lead to compensatory enhancement of antioxidant capacity that protecting leukemia cells from oxidative damage, thereby bringing leukemia cells survival benefits [8, 50]. An example of an oxidative stress signaling-triggered mechanism of protection is regulating nuclear translocation of redox sensitivity transcription factors, e.g., FOXO, nuclear factor (erythroid-derived)-like 2 (Nrf2), hypoxia inducible factors (HIFs), Kelch-like ECH-associated protein 1 (Keap1) etc. Activation of these transcription factors can promote the transcriptional activation of antioxidant defense genes and enhance the cells’ antioxidant capacity to avoid overproduction of ROS [28, 51–53] (Table 1). Interestingly, FOXO can set off the antioxidant system to promote the survival of AML cells and also enhance apoptosis. However, in FLT3-ITD AML cells, the pro-apoptotic effect of FOXO is inhibited by Akt [54] (Fig. 2).Table 1 The main ROS-dependent transcription factors and antioxidant proteins in AML.

Transcription factor/Antioxidant protein	Function	Ref.	
ATM	Promoting antioxidant response through activation of glucose-6-phosphate dehydrogenase (G6PD)	[77]	
FOXOs	Inhibiting ROS generation by upregulating the transcription of antioxidant genes (e.g., ATM) and antioxidant enzymes (e.g., SOD and CAT)	[78]	
HIFs	Protecting AML cells via inhibiting the generation of ROS	[79]	
HO-1	Under oxidative stress, HO-1 expression is induced by the NRF2 transcription factor as part of the cellular antioxidant defense response	[29]	
NF-κB	Regulator of genes involved in regulating the ROS level in cells (e.g., Nrf2)	[38, 80]	
Nrf2	Mediating cytoprotective antioxidant responses via upregulating the transcription of antioxidant genes (e.g., HO-1)	[8, 81]	
Prdx	Protecting intracellular genomic DNA, lipids and proteins from oxidative damage by regulating intracellular ROS levels	[82]	
Trx	Maintaining intracellular redox homeostasis, reducing oxidized protein levels and protein-refolding activity to limit damage caused by oxidative stress	[83]	
ATM Ataxia telangiectasia mutated, FOXO Forkhead box O, HIF hypoxiainducible factor, HO-1 Heme oxygenase-1, NF-κB nuclear factor-kappa, Nrf2 NF-E2-related factor 2, Prdx peroxiredoxin, Trx thioredoxin.

Fig. 2 Redox balance and survival maintenance signaling in FLT3-ITD AML.

FLT3-ITD signaling can upregulate the expression of key DDR factors and MMR factors through the STAT5 and ERK pathways, respectively [10]; The nuclear translocation of redox sensitivity transcription factors, such as Nrf2, NF-κB, and FOXO, etc. can be promoted by ROS, which promote the transcriptional activation of antioxidant defense genes and enhance the antioxidant capacity of cells [28, 51–53]; FOXO can promote the transcription of HDAC8, and then inhibit the antitumor activity of p53 via deacetylation [94]; FOXO also has pro-apoptotic effect, but this effect is inhibited by AKT, whereas the pro-survival effect of BCL-2 and MCL-1 is activated [35, 58–60]. This figure was drawn based on existing research data; its accuracy and more precise signaling mechanisms must be confirmed and supplemented by extensive, in-depth studies.

A commonly accepted chemoresistance mechanism is the oncogenic kinases-driven DNA damage repair activity enhancement which favors the survival of malignant cells under genotoxic stress [55]. Recently, Wu et al. reported that in FLT3-ITD-mutated AML, FLT3-ITD signaling can upregulate the expression of key DNA damage response (DDR) factors, e.g, Wee1-like protein kinase (WEE1), checkpoint kinase 1 (CHK1), proviral integration site for Moloney murine leukemia virus-1 (PIM-1), radiation sensitive 51 (RAD51), and mismatch repair (MMR) factors, e.g, MutS homolog 2 (MSH2), MSH6 and MutL homolog 1 (MLH1) through the STAT5 and ERK pathways, respectively (Fig. 2). These responses could promote AML cell survival under oxidative stress stimulation and induce chemoresistance [10]. In a FLT3-ITD AML mouse model, animals treated with the MK8776 (CHK1 inhibitor) and cytarabine survived longer than those treated with cytarabine alone, suggesting that the combination of DNA damage response inhibitors and conventional chemotherapy may be useful in the treatment of FLT3-ITD AML [10].

Previous studies have shown that suppression of apoptosis signals can also help AML cells maintain survival under oxidative stress. Hole et al. reported that AML cells can escape the oxidative damage by inhibiting p38MAPK-mediated cycle arrest and/or apoptotic signaling [27]. Besides, it was found that the expression or activity of pro-apoptotic proteins, e.g., B-cell lymphoma 2 (Bcl-2) antiagonist of cell death (Bad) and Bcl-2-associated X protein (Bax) are inhibited in FLT3-ITD AML cells [56, 57], whereas the expression or activity of pro-survival proteins, e.g., B-cell lymphoma extra large (Bcl-xL), Bcl-2 or myeloid cell leukemia-1 (MCL-1) are upregulated, which is conducive to AML cells’ survival [35, 58–60]. Therefore, inhibiting the antioxidant capacity of leukemia cells, amplifying oxidative stress signaling, and restoring/activating apoptotic pathways may help to eradicate AML cells [61–65].

Blockade of FLT3-ITD signaling

As the FLT3-ITD oncogene signal is a crucial player in the development of AML, it has emerged as a relevant target in the management of this malignancy. To date, several FLT3 inhibitors have been developed, tested in randomized clinical trials, i.e., midostaurin, gilteritinib, quizartinib, and other TKIs, and approved for clinical application. On this basis, the development of TKIs with multi-target inhibition has been continuously strengthened to improve their efficiency and durability on mutant FLT3 kinases (Table 2). Furthermore, the search for new therapeutic targets is also ongoing. Recent studies have demonstrated that activating mutations in FLT3 can induce an unstable conformation of the kinase, leading to its high dependence on chaperones, e.g., heat shock protein (HSP) 90. Therefore, FLT3-ITD is highly sensitive to the action of HSP90 inhibitors, and treatment with HSP90 inhibitors such as 17-AAG and LAM-003 could result in FLT3-ITD degradation [22, 66]. As NOX is the major source of ROS in FLT3-ITD AML, it is also an ideal target to inhibit FLT3-ITD pro-survival signaling. In FLT3-ITD AML cells, NOX2 inhibitors have been reported to cause decreased intracellular concentrations of ROS, inhibiting FLT3’s growth and survival downstream pathways, and increasing apoptosis associated with restoration of p38MAPK and induction of mitochondrial ROS [67]. Downregulation of NOX4 expression by RNA interference or treatment with NOX4 inhibitors such as schisandrin B also notably decreased ROS production in FLT3-ITD-mutated AML, and reduced both in vitro and in vivo FLT3-ITD-driven signaling and cell transformation [33].Table 2 Novel TKIs in the study of FLT3-ITD AML treatment.

Drug	Description	Function	Ref.	
AMG 925	A potent, selective, and bioavailable FLT3/CDK4 dual kinase inhibitor	Inhibits FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib)	[84]	
Cabozantinib	A multi-targeted TKI of FLT3, MET, AXL, VEGFR, and KIT	Inhibits FLT3-ITD tyrosine kinase in a potent and sustained fashion	[85]	
EC-70124	A hybrid indolocarbazole analog with a potent and selective inhibitory effect on FLT3	Potently inhibits wild-type and mutant FLT3, and also other important kinases such as PIM kinases	[86]	
FN-1501	A potent inhibitor of FLT3 and other tyrosine kinases such as CDK4/6, KIT, PDGFR, ALK and RET tyrosine kinase proteins	Antiproliferative activities against FLT3-ITD expressing cell line MV4-11	[87]	
HM43239	A active small molecule inhibitor of FLT3-ITD, FLT3-TKD, and FLT3- ITD/TKD double mutations	Overcomes the FL-induced drug resistance with a higher cytotoxic potency in MOLM-14 cells harboring FLT3-ITD	[88]	
MZH29	A type II FLT3 inhibitor that tolerated the F691L mutation	Showed sustained inhibitory effects on wild-type and mutant FLT3, including the FLT3-ITD, FLT3-D835H/Y/V and FLT3-K663Q mutants	[89]	
Pexidartinib	A small molecule TKI with selective activity against the CSF1 receptor, KIT and FLT3-ITD	Having inhibitory activity against the FLT3 TKI-resistant F691L gatekeeper mutation in relapsed/refractory FLT3-ITD-mutant AML	[90]	
SEL24	A dual PIM and FLT3-ITD inhibitor	Significant inhibitory activity on FLT3-ITD and tyrosine kinase domain (TKD) positive AML	[91]	
SKLB-677	An FLT3 and Wnt/β-catenin signaling inhibitor	Showed considerable suppression effects on leukemia stem-like cells in in vitro functional assays, but had no influence on normal HSCs	[92]	
ALK anaplastic lymphoma kinase, CDK cyclin-dependent kinase, CSF1 colony-stimulating factor 1, KIT KIT proto-oncogene receptor tyrosine kinase, PDGFR platelet-derived growth factor receptor, R/R relapsed/refractory, RET rearranged during transfection, Wnt, β-catenin.

Protein post-translational modifications (PTMs) are important in the regulation of protein function. As mentioned above, PTMs such as glycosylation and palmitoylation not only affect the subcellular localization of the FLT3-ITD protein, but also lead to alterations in downstream pathways, which may further lead to drug resistance. Therefore, targeting the PTMs of FLT3-ITD protein to overcome drug resistance of AML cells has become a hot topic of current research. According to Williams et al., a lipophilic statin, i.e., fluvastatin, can decrease the mutant FLT3’s kinase activity via preventing advanced glycosylation of the receptor, reducing FLT3-ITD cell surface expression, inhibiting the activation of MAPK and AKT, and reducing the TKI drug resistances while leading to the apoptosis of FLT3-ITD-mutated AML cells [68]. The antineoplastic pharmacological agents 2-deoxy-d-glucose (2-DG) [69] and tunicamycin [70] have also been found to exert pro-apoptotic actions via glycosylation inhibition, as well as FLT3-ITD cell surface expression and signaling. Moreover, the co-administration of FLT3-ITD inhibitors and tunicamycin enhanced cell death synergistically, in particular in FLT3-ITD-mutated AML cells [70]. Recently, an investigation conducted by Lv et al. discovered that palmostatin B, a pan-depalmitoylase inhibitor, dose-dependently inhibited FLT3-ITD cell surface expression, as well as AKT/ERK activation in FLT3-ITD-mutated AML, thereby inhibiting cell growth. Moreover, survival of FLT3-ITD-mutated AML cells was suppressed by the synergism between gilteritinib, a FLT3 kinase inhibitor, and depalmitoylation inhibitors. Thus, depalmitoylation may emerge as a future therapeutic target in FLT3-ITD-mutated AML [19].

Blockage of intracellular transport routes of FLT3-ITD has also been shown to effectively inhibit oncogenic signals. Yamawaki et al. reported that the use of 2-methylcoprophilinamide (M-COPA) or brefeldin A to inhibit FLT3-ITD’s export from the ER or the use of monensin to inhibit FLT3-ITD’s export from the Golgi apparatus can notably decrease signals of tyrosine phosphorylation in AML [21]. In addition, according to Moloney et al., retention of FLT3-ITD in the ER can occur following treatment with brefeldin A and tunicamycin, leading to reduced NOX4 and p22phox – the partner protein of NOX4—expression. Consequently, the generation of pro-survival ROS is blocked [40]. As FLT3-ITD is a misfolded protein, it is noteworthy to mention that the massive accumulation in the ER of misfolded proteins leads to increased cellular susceptibility to ER stress and oxidative stress [71]. As reported by Masciarelli et al., a combination therapy consisting of low concentrations of retinoic acid, tunicamycin, and arsenic trioxide can induce oxidative stress and ER stress, resulting in FLT3-ITD-mutated AML cells’ apoptosis. Based on the aforementioned findings, it seems that AML cells are sensitive to ER stress and oxidative stress which may lay the path for novel therapeutic targets to eradicate this hematological malignancy [71].

The strategy of blocking the downstream signal of FLT3-ITD has also attracted much attention. For example, the oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and stabilizes and phosphorylates FLT3-ITD, thus promoting the signaling of FLT3-ITD in a positive feedback loop in AML cells [72]. Baldwin et al. explored the synergism between quizartinib (FLT3 inhibitor) and azd1208 (PIM inhibitor) in FLT3-ITD-mutated AML. As compared to their singular use, the combined use of these two pharmacological agents showed synergistic effect and more efficiency in inhibiting AML cells’ growth and enhanced their apoptosis both in vivo and in vitro. The explanation behind this result could be that this drug combination downregulates the anti-apoptotic protein Mcl-1’s expression [72]. According to Jasek Gajda et al., another combination of pharmacological agents, namely ZSTK474 (PI3K inhibitor) and AZD0364 (ERK1/2 inhibitor) can suppress the in vitro activation of Akt and ERK1/2 in molm-14 cells, i.e., in a cellular model of FLT3-ITD-mutated AML, induce the apoptosis of AML cells by promoting the generation of ROS [48]. In vivo and in vitro experiments have confirmed that AC-4-130, an inhibitor of the SH2-domain of STAT5, is able to suppress the pathological activity of STAT5 in FLT3-ITD-mutated AML, thereby effectively inhibiting the proliferation of AML cells [73].

Conclusions and future perspectives

The FLT3-ITD mutation is one of the most important gene mutations in AML, and it is also a crucial indicator of poor prognosis in this blood cancer. Current second-generation TKIs, such as sorafenib, gilteritinib, crenolanib, and quizartinib, are highly specific and potent, and often lead to favorable outcomes when employed in the early treatment of FLT3-ITD-mutated AML. However, the efficacy of TKIs is often challenged by the development of resistance caused by a myriad of factors, e.g., secondary mutations or signaling compensations. Therefore, most of these drugs fail to achieve the expected efficacy.

Recently, a global untargeted metabolomics and stable isotope-labeling mass spectrometry analysis was applied to evaluate sorafenib resistance in FLT3-ITD-mutated leukemic cells [50]. It was shown that resistant cells display fundamentally rewired metabolic profiles, characterized by elevated need for glucose, accompanied by a reduction in glucose flux into the pentose phosphate pathway. Besides, increased oxidative stress was accompanied by an enhanced antioxidant capacity of glutathione. These results demonstrate that FLT3-ITD-mutated leukemia cells have obvious metabolic and redox adaptability which may contribute to the development of sorafenib resistance [50]. Based on transcriptomic analyses, Zavorka Thomas et al. identified glutamine transporter SNAT1 (SLC38A1) as a novel target for gilteritinib. In addition, it was highlighted that impaired glutamine uptake and utilization within leukemic cells can enhance the efficacy of gilteritinib [74]. Similarly, another FLT3 inhibitor, namely AC220, causes impaired glutamine metabolism in AML, particularly when co-administered with CB-839, i.e., an inhibitor of glutaminase, leading to elevated ROS production in the mitochondria of leukemic cells and depleting their glutathione sources. Thus, this treatment promotes cell death in this blood cancer, suggesting new therapeutical strategies for a subset of relapsed/refractory AML [75].

The use of antioxidants during leukemia therapy is also currently a hot topic. It was recently reported that N-acetyl-cysteine, a natural product, was able to reduce ROS generation in FLT3-ITD-mutated AML and contributed to the prevention of aberrant interchromosomal homologous recombinations which are involved in leukemia survival and progression [76]. Theoretically, the use of antioxidants may help reduce TKI resistance caused by secondary mutations. However, antioxidants will inevitably weaken the therapeutic effect of pro-oxidant chemotherapy drugs. Therefore, exploiting new antioxidants with synergistic anti-leukemia effects may aid in providing solutions to this unsolved dilemma.

Author contributions

Conceptualization, YC and JL; Writing—original draft preparation, YC, JL and M-AG; Writing—review and editing, LX, ZZ and M-AG; Prepared figures: YC and JL; Supervision, YC and M-AG. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the scientific research project of the Sichuan Province Education Department (No. 16ZA0241), the National Natural Science Foundation of China [82060268], and the Guangxi Natural Science Foundation of China [2020JJA140124].

Competing interests

The authors declare no competing interests.

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

These authors contributed equally: Yongfeng Chen, Zhenyou Zou.
==== Refs
References

1. Crawford LB Kim JH Collins-McMillen D Lee BJ Landais I Held C Human cytomegalovirus encodes a novel FLT3 receptor ligand necessary for hematopoietic cell differentiation and viral reactivation mBio. 2018 9 e00682 18. 10.1128/mBio.00682-18 29691342
2. Safaei A Monabati A Mokhtari M Safavi M Solhjoo F Evaluation of the CD123 expression and FLT3 gene mutations in patients with acute myeloid leukemia Iran J Pathol 2018 13 438 46 30774683
3. Kaweme NM Zhou S Changwe GJ Zhou F The significant role of redox system in myeloid leukemia: from pathogenesis to therapeutic applications Biomark Res 2020 8 63 10.1186/s40364-020-00242-z 33292641
4. Kazi JU Rönnstrand L FMS-like tyrosine kinase 3/FLT3: from basic science to clinical implications Physiol Rev 2019 99 1433 66. 10.1152/physrev.00029.2018 31066629
5. Cai J Huang H Hu X Lang W Fu W Xu L Homoharringtonine synergized with gilteritinib results in the downregulation of myeloid cell leukemia-1 by upregulating UBE2L6 in FLT3-ITD-mutant acute myeloid (leukemia) cell lines J Oncol 2021 2021 3766428 10.1155/2021/3766428 34594375
6. Schmalbrock LK Dolnik A Cocciardi S Sträng E Theis F Jahn N Clonal evolution of acute myeloid leukemia with FLT3-ITD mutation under treatment with midostaurin Blood. 2021 137 3093 104 10.1182/blood.2020007626 33598693
7. Tazi Y Arango-Ossa JE Zhou Y Bernard E Thomas I Gilkes A Unified classification and risk-stratification in Acute Myeloid Leukemia Nat Commun 2022 13 4622 10.1038/s41467-022-32103-8 35941135
8. Sallmyr A Fan J Datta K Kim KT Grosu D Shapiro P Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML Blood. 2008 111 3173 82 10.1182/blood-2007-05-092510 18192505
9. Germon ZP Sillar JR Mannan A Duchatel RJ Staudt D Murray HC Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies Sci Signal 2023 16 eabp9586 10.1126/scisignal.abp9586 36976863
10. Wu M Li L Hamaker M Small D Duffield AS FLT3-ITD cooperates with Rac1 to modulate the sensitivity of leukemic cells to chemotherapeutic agents via regulation of DNA repair pathways Haematologica. 2019 104 2418 28 10.3324/haematol.2018.208843 30975911
11. Moloney JN Stanicka J Cotter TG Subcellular localization of the FLT3-ITD oncogene plays a significant role in the production of NOX- and p22phox-derived reactive oxygen species in acute myeloid leukemia Leuk Res 2017 52 34 42 10.1016/j.leukres.2016.11.006 27870947
12. Stanicka J Russell EG Woolley JF Cotter TG NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells J Biol Chem 2015 290 9348 61 10.1074/jbc.M113.510495 25697362
13. Chen YF Liu H Luo XJ Zhao Z Zou ZY Li J The roles of reactive oxygen species (ROS) and autophagy in the survival and death of leukemia cells Crit Rev Oncol Hematol 2017 112 21 30 10.1016/j.critrevonc.2017.02.004 28325262
14. Lagadinou ED Sach A Callahan K Rossi RM Neering SJ Minhajuddin M BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells Cell Stem Cell 2013 12 329 41 10.1016/j.stem.2012.12.013 23333149
15. Vetrie D Helgason GV Copland M The leukaemia stem cell: similarities, differences and clinical prospects in CML and AML Nat Rev Cancer 2020 20 158 73. 10.1038/s41568-019-0230-9 31907378
16. Rebechi MT Pratz KW Genomic instability is a principle pathologic feature of FLT3 ITD kinase activity in acute myeloid leukemia leading to clonal evolution and disease progression Leuk Lymphoma 2017 58 1 11 10.1080/10428194.2017.1283031 28278729
17. Müller JP Schmidt-Arras D Novel approaches to target mutant FLT3 leukaemia Cancers (Basel) 2020 12 2806 10.3390/cancers12102806 33003568
18. Schmidt-Arras DE Böhmer A Markova B Choudhary C Serve H Böhmer FD Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases Mol Cell Biol 2005 25 3690 703 10.1128/MCB.25.9.3690-3703.2005 15831474
19. Lv K Ren JG Han X Gui J Gong C Tong W Depalmitoylation rewires FLT3-ITD signaling and exacerbates leukemia progression Blood 2021 138 2244 55. 10.1182/blood.2021011582 34111291
20. Choudhary C Olsen JV Brandts C Cox J Reddy PN Böhmer FD Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes Mol Cell 2009 36 326 39 10.1016/j.molcel.2009.09.019 19854140
21. Yamawaki K Shiina I Murata T Tateyama S Maekawa Y Niwa M FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells Sci Rep. 2021 11 22678 10.1038/s41598-021-02221-2 34811450
22. Fleischmann M Fischer M Schnetzke U Fortner C Kirkpatrick J Heidel FH Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance Cells. 2021 10 2992 10.3390/cells10112992 34831215
23. Wang Y Xu Y Li S Liu J Xing Y Xing H Targeting FLT3 in acute myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells J Hematol Oncol 2018 11 60 10.1186/s13045-018-0603-7 29716633
24. Chen YF Li J Xu LL Găman MA Zou ZY Allogeneic stem cell transplantation in the treatment of acute myeloid leukemia: an overview of obstacles and opportunities World J Clin Cases 2023 11 268 91 10.12998/wjcc.v11.i2.268 36686358
25. Ju HQ Zhan G Huang A Sun Y Wen S Yang J ITD mutation in FLT3 tyrosine kinase promotes Warburg effect and renders therapeutic sensitivity to glycolytic inhibition Leukemia. 2017 31 2143 50. 10.1038/leu.2017.45 28194038
26. Erdem A Marin S Pereira-Martins DA Geugien M Cunningham A Pruis MG Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability Nat Commun 2022 13 2013 10.1038/s41467-022-29639-0 35440568
27. Hole PS Zabkiewicz J Munje C Newton Z Pearn L White P Overproduction of NOX-derived ROS in AML promotes proliferation and is associated with defective oxidative stress signaling Blood. 2013 122 3322 30 10.1182/blood-2013-04-491944 24089327
28. Trombetti S Cesaro E Catapano R Sessa R Lo Bianco A Izzo P Oxidative stress and ROS-mediated signaling in leukemia: novel promising perspectives to eradicate chemoresistant cells in myeloid leukemia Int J Mol Sci 2021 22 2470 10.3390/ijms22052470 33671113
29. Prieto-Bermejo R Romo-González M Pérez-Fernández A Ijurko C Hernández-Hernández Á Reactive oxygen species in haematopoiesis: leukaemic cells take a walk on the wild side J Exp Clin Cancer Res 2018 37 125 10.1186/s13046-018-0797-0 29940987
30. Su H Guan G Ahmed RZ Lyu L Li Z Jin X TBBPA stimulated cell migration of endometrial cancer via the contribution of NOX-generated ROS in lieu of energy metabolism J Hazard Mater 2020 400 123204 10.1016/j.jhazmat.2020.123204 32569978
31. Hole PS Darley RL Tonks A Do reactive oxygen species play a role in myeloid leukemias? Blood. 2011 117 5816 26 10.1182/blood-2011-01-326025 21398578
32. Brault J Vigne B Meunier M Beaumel S Mollin M Park S NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells Free Radic Biol Med 2020 146 107 18. 10.1016/j.freeradbiomed.2019.10.005 31626946
33. Jayavelu AK Müller JP Bauer R Böhmer SA Lässig J Cerny-Reiterer S NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells Leukemia. 2016 30 473 83 10.1038/leu.2015.234 26308771
34. Darici S Alkhaldi H Horne G Jørgensen HG Marmiroli S Huang X Targeting PI3K/Akt/mTOR in AML: rationale and clinical evidence J Clin Med 2020 9 2934 10.3390/jcm9092934 32932888
35. Zhang Q Riley-Gillis B Han L Jia Y Lodi A Zhang H Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia Signal Transduct Target Ther 2022 7 51 10.1038/s41392-021-00870-3 35185150
36. Anstee NS Bilardi RA Ng AP Xu Z Robati M Vandenberg CJ Impact of elevated anti-apoptotic MCL-1 and BCL-2 on the development and treatment of MLL-AF9 AML in mice Cell Death Differ 2019 26 1316 31. 10.1038/s41418-018-0209-1 30470795
37. Yuan F Li D Li G Cheng C Wei X Synergistic efficacy of homoharringtonine and venetoclax on acute myeloid leukemia cells and the underlying mechanisms Ann Transl Med 2022 10 490 10.21037/atm-22-1459 35571387
38. Samimi A Kalantari H Lorestani MZ Shirzad R Saki N Oxidative stress in normal hematopoietic stem cells and leukemia APMIS. 2018 126 284 94. 10.1111/apm.12822 29575200
39. Adachi Y Shibai Y Mitsushita J Shang WH Hirose K Kamata T Oncogenic Ras upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphorylation of GATA-6 Oncogene. 2008 27 4921 32 10.1038/onc.2008.133 18454176
40. Moloney JN Jayavelu AK Stanicka J Roche SL O'Brien RL Scholl S Nuclear membrane-localised NOX4D generates pro-survival ROS in FLT3-ITD-expressing AML Oncotarget. 2017 8 105440 57. 10.18632/oncotarget.22241 29285262
41. Hwang DY Eom JI Jang JE Jeung HK Chung H Kim JS ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia J Exp Clin Cancer Res 2020 39 85 10.1186/s13046-020-01580-4 32393312
42. Rummelt C Gorantla SP Meggendorfer M Charlet A Endres C Döhner K Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo Leukemia. 2021 35 2017 29 10.1038/s41375-020-01077-1 33149267
43. Zhang B Ayuda-Durán P Piechaczyk L Fløisand Y Safont MM Karlsen IT GRP94 rewires and buffers the FLT3-ITD signaling network and promotes survival of acute myeloid leukemic stem cells Haematologica. 2019 104 e229 10.3324/haematol.2019.220533 31040234
44. Smith CC Viny AD Massi E Kandoth C Socci ND Rapaport F Recurrent mutations in cyclin D3 confer clinical resistance to FLT3 inhibitors in acute myeloid leukemia Clin Cancer Res 2021 27 4003 11 10.1158/1078-0432.CCR-20-3458 34103301
45. Waldeck S Rassner M Keye P Follo M Herchenbach D Endres C CCL5 mediates target-kinase independent resistance to FLT3 inhibitors in FLT3-ITD-positive AML Mol Oncol 2020 14 779 94. 10.1002/1878-0261.12640 31955503
46. Godfrey R Arora D Bauer R Stopp S Müller JP Heinrich T Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ Blood. 2012 119 4499 511 10.1182/blood-2011-02-336446 22438257
47. Arora D Stopp S Böhmer SA Schons J Godfrey R Masson K Protein-tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling J Biol Chem 2011 286 10918 29 10.1074/jbc.M110.205021 21262971
48. Jasek-Gajda E Jurkowska H Jasińska M Lis GJ Targeting the MAPK/ERK and PI3K/AKT signaling pathways affects NRF2, Trx and GSH antioxidant systems in leukemia cells Antioxid (Basel) 2020 9 633 10.3390/antiox9070633
49. Chen Y Li J Zhao Z Redox control in acute lymphoblastic leukemia: from physiology to pathology and therapeutic opportunities Cells 2021 10 1218 10.3390/cells10051218 34067520
50. You X Jiang W Lu W Zhang H Yu T Tian J Metabolic reprogramming and redox adaptation in sorafenib-resistant leukemia cells: detected by untargeted metabolomics and stable isotope tracing analysis Cancer Commun (Lond) 2019 39 17 10.1186/s40880-019-0362-z 30947742
51. Darwish NHE Sudha T Godugu K Bharali DJ Elbaz O El-Ghaffar HAA Novel targeted nano-parthenolide molecule against NF-kB in acute myeloid leukemia Molecules. 2019 24 2103 10.3390/molecules24112103 31163672
52. Hoang DH Buettner R Valerio M Ghoda L Zhang B Kuo YH Arsenic trioxide and venetoclax synergize against AML progenitors by ROS induction and inhibition of Nrf2 activation Int J Mol Sci 2022 23 6568 10.3390/ijms23126568 35743010
53. Chen Y Liang Y Luo X Hu Q Oxidative resistance of leukemic stem cells and oxidative damage to hematopoietic stem cells under pro-oxidative therapy Cell Death Dis 2020 11 291 10.1038/s41419-020-2488-y 32341354
54. Brandts CH Sargin B Rode M Biermann C Lindtner B Schwäble J Constitutive activation of Akt by Flt3 internal tandem duplications is necessary for increased survival, proliferation, and myeloid transformation Cancer Res 2005 65 9643 50 10.1158/0008-5472.CAN-05-0422 16266983
55. Zhang J Gu Y Chen B Mechanisms of drug resistance in acute myeloid leukemia Onco Targets Ther 2019 12 1937 45 10.2147/OTT.S191621 30881045
56. Sobhanifar MA Mashkani B Saadatmandzadeh M Sadeghnia HR Mousavi SH Induction of cytotoxicity and apoptosis in FLT3 mutant expressing cells using novel pyrimido cyanoacrylates and quinoline derivatives Biomed Pharmacother 2018 108 893 905 10.1016/j.biopha.2018.09.001 30372901
57. Kim KT Levis M Small D Constitutively activated FLT3 phosphorylates BAD partially through pim-1 Br J Haematol 2006 134 500 9 10.1111/j.1365-2141.2006.06225.x 16869825
58. Singh Mali R Zhang Q DeFilippis RA Cavazos A Kuruvilla VM Raman J Venetoclax combines synergistically with FLT3 inhibition to effectively target leukemic cells in FLT3-ITD+ acute myeloid leukemia models Haematologica. 2021 106 1034 46 10.3324/haematol.2019.244020 32414851
59. Zhu R Li L Nguyen B Seo J Wu M Seale T FLT3 tyrosine kinase inhibitors synergize with BCL-2 inhibition to eliminate FLT3/ITD acute leukemia cells through BIM activation Signal Transduct Target Ther 2021 6 186 10.1038/s41392-021-00578-4 34024909
60. Nogami A Okada K Ishida S Akiyama H Umezawa Y Miura O Inhibition of the STAT5/Pim kinase axis enhances cytotoxic effects of proteasome inhibitors on FLT3-ITD-positive AML cells by cooperatively inhibiting the mTORC1/4EBP1/S6K/Mcl-1 pathway Transl Oncol 2019 12 336 49. 10.1016/j.tranon.2018.11.001 30472492
61. Hsiao PC Chang JH Lee WJ Ku CC Tsai MY Yang SF The curcumin analogue, EF-24, triggers p38 MAPK-mediated apoptotic cell death via inducing PP2A-modulated ERK deactivation in human acute myeloid leukemia cells Cancers (Basel) 2020 12 2163 10.3390/cancers12082163 32759757
62. Hosseini M Rezvani HR Aroua N Bosc C Farge T Saland E Targeting myeloperoxidase disrupts mitochondrial redox balance and overcomes cytarabine resistance in human acute myeloid leukemia Cancer Res 2019 79 5191 203. 10.1158/0008-5472.CAN-19-0515 31358527
63. Yang Y Wang G Wu W Yao S Han X He D Camalexin induces apoptosis via the ROS-ER stress-mitochondrial apoptosis pathway in AML cells Oxid Med Cell Longev 2018 2018 7426950 10.1155/2018/7426950 30538806
64. Chen Y Luo X Zou Z Liang Y The role of reactive oxygen species in tumor treatment and its impact on bone marrow hematopoiesis Curr Drug Targets 2020 21 477 98 10.2174/1389450120666191021110208 31736443
65. Chen Y Li J Xu L Găman MA Zou Z The genesis and evolution of acute myeloid leukemia stem cells in the microenvironment: From biology to therapeutic targeting Cell Death Discov 2022 8 397 10.1038/s41420-022-01193-0 36163119
66. Beeharry N Landrette S Gayle S Hernandez M Grotzke JE Young PR LAM-003, a new drug for treatment of tyrosine kinase inhibitor-resistant FLT3-ITD-positive AML Blood Adv 2019 3 3661 73. 10.1182/bloodadvances.2019001068 31751472
67. Germon ZP, Sillar JR, Mannan A, Duchatel RJ, Staudt D, Murray HC, et al. Blockade of redox second messengers inhibits JAK/STAT and MEK/ERK signaling sensitizing FLT3-mutant acute myeloid leukemia to targeted therapies. bioRxiv. 2022;2022-03. https://www.biorxiv.org/content/10.1101/2022.03.09.483687v1.
68. Williams AB Li L Nguyen B Brown P Levis M Small D Fluvastatin inhibits FLT3 glycosylation in human and murine cells and prolongs survival of mice with FLT3/ITD leukemia Blood 2012 120 3069 79 10.1182/blood-2012-01-403493 22927251
69. Larrue C Saland E Vergez F Serhan N Delabesse E Mansat-De Mas V Antileukemic activity of 2-deoxy-d-glucose through inhibition of N-linked glycosylation in acute myeloid leukemia with FLT3-ITD or c-KIT mutations Mol Cancer Ther 2015 14 2364 73 10.1158/1535-7163.MCT-15-0163 26206337
70. Tsitsipatis D Jayavelu AK Müller JP Bauer R Schmidt-Arras D Mahboobi S Synergistic killing of FLT3ITD-positive AML cells by combined inhibition of tyrosine-kinase activity and N-glycosylation Oncotarget 2017 8 26613 24. 10.18632/oncotarget.15772 28460451
71. Masciarelli S Capuano E Ottone T Divona M Lavorgna S Liccardo F Retinoic acid synergizes with the unfolded protein response and oxidative stress to induce cell death in FLT3-ITD+ AML Blood Adv 2019 3 4155 60. 10.1182/bloodadvances.2019000540 31834935
72. Baldwin PR Kapoor S Natarajan K Trotta R Tron A Huszar D Concurrent inhibition of Pim-1 and FLT3 kinases in FLT3-ITD acute myeloid leukemia post-translationally downregulates the anti-apoptotic protein Mcl-1 through downregulation of the Mcl-1 deubiquitinase USP9X Blood 2016 128 35 10.1182/blood.V128.22.35.35
73. Wingelhofer B Maurer B Heyes EC Cumaraswamy AA Berger-Becvar A de Araujo ED Pharmacologic inhibition of STAT5 in acute myeloid leukemia Leukemia. 2018 32 1135 46. 10.1038/s41375-017-0005-9 29472718
74. Zavorka Thomas ME Lu X Talebi Z Jeon JY Buelow DR Gibson AA Gilteritinib inhibits glutamine uptake and utilization in FLT3-ITD-positive AML Mol Cancer Ther 2021 20 2207 17 10.1158/1535-7163.MCT-21-0071 34518298
75. Gregory MA Nemkov T Reisz JA Zaberezhnyy V Hansen KC D'Alessandro A Glutaminase inhibition improves FLT3 inhibitor therapy for acute myeloid leukemia Exp Hematol 2018 58 52 58 10.1016/j.exphem.2017.09.007 28947392
76. Gaymes TJ Mohamedali A Eiliazadeh AL Darling D Mufti GJ FLT3 and JAK2 mutations in acute myeloid leukemia promote interchromosomal homologous recombination and the potential for copy neutral loss of heterozygosity Cancer Res 2017 77 1697 708 10.1158/0008-5472.CAN-16-1678 28108507
77. Gregory MA D'Alessandro A Alvarez-Calderon F Kim J Nemkov T Adane B ATM/G6PD-driven redox metabolism promotes FLT3 inhibitor resistance in acute myeloid leukemia Proc Natl Acad Sci USA 2016 113 E6669 E6678 10.1073/pnas.1603876113 27791036
78. Richardson C Yan S Vestal CG Oxidative stress, bone marrow failure, and genome instability in hematopoietic stem cells Int J Mol Sci 2015 16 2366 85 10.3390/ijms16022366 25622253
79. Rouault-Pierre K Lopez-Onieva L Foster K Anjos-Afonso F Lamrissi-Garcia I Serrano-Sanchez M HIF-2α protects human hematopoietic stem/progenitors and acute myeloid leukemic cells from apoptosis induced by endoplasmic reticulum stress Cell Stem Cell 2013 13 549 63 10.1016/j.stem.2013.08.011 24095676
80. Rushworth SA Zaitseva L Murray MY Shah NM Bowles KM MacEwan DJ The high Nrf2 expression in human acute myeloid leukemia is driven by NF-κB and underlies its chemo-resistance Blood. 2012 120 5188 98 10.1182/blood-2012-04-422121 23077289
81. Xu B Wang S Li R Chen K He L Deng M Disulfiram/copper selectively eradicates AML leukemia stem cells in vitro and in vivo by simultaneous induction of ROS-JNK and inhibition of NF-κB and Nrf2 Cell Death Dis 2017 8 e2797 10.1038/cddis.2017.176 28518151
82. Jin Y Yang Q Liang L Ding L Liang Y Zhang D Compound kushen injection suppresses human acute myeloid leukaemia by regulating the Prdxs/ROS/Trx1 signalling pathway J Exp Clin Cancer Res 2018 37 277 10.1186/s13046-018-0948-3 30454068
83. Zhou F Pan Y Wei Y Zhang R Bai G Shen Q Jab1/Csn5-thioredoxin signaling in relapsed acute monocytic leukemia under oxidative stress Clin Cancer Res 2017 23 4450 61. 10.1158/1078-0432.CCR-16-2426 28270496
84. Keegan K Li C Li Z Ma J Ragains M Coberly S Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor for treating acute myeloid leukemia Mol Cancer Ther 2014 13 880 9 10.1158/1535-7163.MCT-13-0858 24526162
85. Fathi AT Blonquist TM Levis MJ Hernandez D Ballen KK Avigan DE A phase I study of the multi-targeted tyrosine kinase inhibitor cabozantinib in patients with acute myeloid leukemia Blood 2016 128 5218 10.1182/blood.V128.22.5218.5218
86. Puente-Moncada N Costales P Antolín I Núñez LE Oro P Hermosilla MA Inhibition of FLT3 and PIM kinases by EC-70124 exerts potent activity in preclinical models of acute myeloid leukemia Mol Cancer Ther 2018 17 614 24. 10.1158/1535-7163.MCT-17-0530 29339551
87. Wang Y Zhi Y Jin Q Lu S Lin G Yuan H Discovery of 4-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (FN-1501), an FLT3- and CDK-kinase inhibitor with potentially high efficiency against acute myelocytic leukemia J Med Chem 2018 61 1499 518. 10.1021/acs.jmedchem.7b01261 29357250
88. Kim JS Bae IH Choi JY HM43239, a novel FLT3 inhibitor in overcoming resistance for acute myeloid leukemia Cancer Res 2019 79 1293 10.1158/1538-7445.AM2019-1293 30936073
89. Xu B Zhao Y Wang X Gong P Ge W MZH29 is a novel potent inhibitor that overcomes drug resistance FLT3 mutations in acute myeloid leukemia Leukemia. 2017 31 913 21. 10.1038/leu.2016.297 27773927
90. Smith CC Levis MJ Frankfurt O Pagel JM Roboz GJ Stone RM A phase 1/2 study of the oral FLT3 inhibitor pexidartinib in relapsed/refractory FLT3-ITD-mutant acute myeloid leukemia Blood Adv 2020 4 1711 21 10.1182/bloodadvances.2020001449 32330242
91. Czardybon W Windak R Gołas A Gałęzowski M Sabiniarz A Dolata I A novel, dual pan-PIM/FLT3 inhibitor SEL24 exhibits broad therapeutic potential in acute myeloid leukemia Oncotarget. 2018 9 16917 31 10.18632/oncotarget.24747 29682194
92. Ma S Yang LL Niu T Cheng C Zhong L Zheng MW SKLB-677, an FLT3 and Wnt/β-catenin signaling inhibitor, displays potent activity in models of FLT3-driven AML Sci Rep. 2015 5 15646 10.1038/srep15646 26497577
93. Ng KL. Targeting DNA damage and repair mechanism in FLT3-ITD acute myeloid leukemia: a mechanistic and therapeutic study[D]. King’s College London, 2018.
94. Long J Jia MY Fang WY Chen XJ Mu LL Wang ZY FLT3 inhibition upregulates HDAC8 via FOXO to inactivate p53 and promote maintenance of FLT3-ITD+ acute myeloid leukemia Blood. 2020 135 1472 83. 10.1182/blood.2019003538 32315388