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FLT3 down-regulation is a specific phenomenon . ( A ) Western blot of whole lysates of Ba/F3 cell line stably transfected with different FLT3 ITD constructs (W51, W78, NPOS) show down-regulation of FLT3 at hypoxic conditions. ( B ) Ba/F3 cells stably transfected with the kinase inactive FLT3 K644R mutant also down-regulated FLT3 under 1% O 2 . ( C ) No regulation of EGF-R by hypoxia of 1% O 2 within 72 hours in Ba/F3 cells. ( D ) Ba/F3 cells stably transfected with Epo-R show no regulation of Epo-R by hypoxia. ( E ) Fold change of FLT3 OD normalized to β -actin in Ba/F3 W51, Ba/F3 W78 and Ba/F3 NPOS cells shows a statistical significance of FLT3 down-regulation under hypoxic conditions, whereas fold change of EGF-r and EPO-r OD do not show a significant down-regulation of this receptors under hypoxia. ( F ) No regulation of EGF-R and KIT receptor in n = 4 AML patient samples previously tested for FLT3 down-regulation under hypoxia (n = 3 down-regulating patients, n = 1 not regulating patient). ( G ) Optical density (OD) of KIT receptor and EGF-r western blot bands normalized to β -actin shows no statistical significance regulation under hypoxia in n = 4 AML patient samples.
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Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenviro...
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Context 1
... is independent from the mutational state of the receptor. To further validate this hypothesis, we investigated the expression of FLT3 at 1% O 2 in 2 additional Ba/F3 FLT3-ITD mutants (Ba/F3 W78 and Ba/F3 NPOS) and in the kinase-inactive Ba/F3 K644R mutant. The hypoxia medi- ated down-regulation was also observed in each of these Ba/F3 cells (Fig. 4A,B). Next, we investigated whether hypoxia-mediated down-regulation is a general phenomenon applying to other tyrosine kinase receptors (TKR) and cytokine receptors as well; especially receptors that have a key role in haemato- poiesis and leukaemogenesis as EGF-R, c-KIT and Epo-R. Ba/F3 cells stably transfected with EGF-R and Epo-R, were ...
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... is a general phenomenon applying to other tyrosine kinase receptors (TKR) and cytokine receptors as well; especially receptors that have a key role in haemato- poiesis and leukaemogenesis as EGF-R, c-KIT and Epo-R. Ba/F3 cells stably transfected with EGF-R and Epo-R, were analysed for a 3-day time kinetic at 21% O 2 and 1% O 2 and, as shown in Fig. 4C,D, we failed to observe any hypoxia-induced regulation of the respective ...
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... levels of EGF-R and KIT receptor were checked in primary samples (n = 4) and results are shown in Fig. 4F: there was no regulation of EGF-R and KIT receptor under 1% O 2 , regardless of FLT3 regulation. In particular, individual differences between different patients were observed for KIT receptor expression under 1% O 2 compared to 21% O 2 , nevertheless no significant down-regulation was observed in any of the patient sample tested (Fig. ...
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... shown in Fig. 4F: there was no regulation of EGF-R and KIT receptor under 1% O 2 , regardless of FLT3 regulation. In particular, individual differences between different patients were observed for KIT receptor expression under 1% O 2 compared to 21% O 2 , nevertheless no significant down-regulation was observed in any of the patient sample tested (Fig. ...
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Acute Myeloid Leukemia (AML) has grave prognosis due to aggressive nature of the disease, the toxicity of standard treatment, and overall low cure rates. We recently showed that AML cells in established culture treated with Cytarabine (AraC) and a differentiation agent combination show enhancement of AraC cytotoxicity. Here we elucidate molecular c...
Citations
... 47 Hypoxia was shown to down-regulate FLT3 and FLT3-ITD signaling in AML cells. 48 Under such conditions, the PI3K/AKT pathway was shown to sustain AML cell survival, rescuing FLT3 activity, 49 while AXL is known to activate PI3K/AKT signaling in AML cells. We and others have previously shown that cytokine-activated STAT5 enhances HIF2α expression directly 44 but also indirectly by inhibiting one of its destabilizers, the prolyl 4-hydroxylase domain protein 3 (PHD3) in both normal hematopoietic progenitors and leukemic cells. ...
... Because so many mechanisms participate in the maintenance of the LIC, the challenge is to inhibit all of them while sparing normal hematopoiesis. FLT3-ITD is no longer expressed in hypoxia, such as inside the bone marrow [9][10][11]. However, it has been detected in the immature CD34 + /CD38-/CD123 + fraction in AML [12,13]. ...
Simple Summary
One of the most frequent molecular anomalies in acute myeloid leukemia (AML) is the mutation of the fms-like receptor tyrosine kinase 3 through internal tandem duplications, giving rise to a constitutive proliferative signaling. Even though clinical trials have shown that targeting this mutated kinase is of interest and well tolerated, there is still a high frequency of relapse. The emergence of AML cells upon treatment is linked to their maintenance through resistance and persistence mechanisms. Because FLT3-ITD AML cells require autophagy, we explored the consequence of autophagy inhibition by blocking the PI3-kinase class III, Vps34, when AML cells were committed. Results in vitro, ex vivo and in vivo suggest that remission with low minimal residual disease in FLT3-ITD AML offers a promising therapeutic window to target persistent leukemic cells.
Abstract
Targeting FLT3-ITD in AML using TKI against FLT3 cannot prevent relapse even in the presence of complete remission, suggesting the resistance and/or the persistence of leukemic-initiating cells in the hematopoietic niche. By mimicking the hematopoietic niche condition with cultures at low oxygen concentrations, we demonstrate in vitro that FLT3-ITD AML cells decrease their repopulating capacity when Vps34 is inhibited. Ex vivo, AML FLT3-ITD blasts treated with Vps34 inhibitors recovered proliferation more slowly due to an increase an apoptosis. In vivo, mice engrafted with FLT3-ITD AML MV4-11 cells have the invasion of the bone marrow and blood in 2 weeks. After 4 weeks of FLT3 TKI treatment with gilteritinib, the leukemic burden had strongly decreased and deep remission was observed. When treatment was discontinued, mice relapsed rapidly. In contrast, Vps34 inhibition strongly decreased the relapse rate, and even more so in association with mobilization by G-CSF and AMD3100. These results demonstrate that remission offers the therapeutic window for a regimen using Vps34 inhibition combined with mobilization to target persistent leukemic stem cells and thus decrease the relapse rate.
... Because so many mechanisms participate in the maintenance of the LIC, the challenge is to inhibit all of them while sparing normal hematopoiesis. FLT3-ITD is no longer expressed in hypoxia, such as inside the bone marrow [9][10][11]. However, it has been detected in the immature CD34 + /CD38-/CD123 + fraction in AML [12,13]. ...
... Results shown in (A) represent the mean ± SEM fold increase in mRNA aggressive forms of blood cancer and carries a dismal prognosis due to short remission durations and high relapse rates.42 The dynamics associated with FLT3/ITD + AML indicate that distinct mechanisms of resistance and MRD are operative in this disease, thus making it an ideal model to study the effects of novel therapeutic approaches.Although established as an effective drug target in AML, previous data has shown that FLT3 is downregulated in response to hypoxic stress,43 indicating that other signalling pathways influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. ...
Acute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O2)-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O2-deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O2 conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O2-deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.
... Wang et al. 2016), which is advantageous for their functionality.However, at the same time hypoxia can decrease CART-cells proliferation and hinder their effector memory differentiation and functionality(Rodriguez-Garcia et al. 2020). In another hand, hypoxic environment can likely induce alteration of the surface phenotype of AML cells(Sironi et al. 2015) ...
Acute myeloid leukemia (AML) is globally a rare disease affecting more commonly older adults and among men compared to women. The rate of new cases of AML was 4.3 per 100,000 men and women per year. AML remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are a subpopulation of AML cells with self-renewal, and chemorefractory capacity. AML LSCs are the basis of refractory/relapsed (R/R) disease in 80% of AML patients not receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). Treatment of relapsed and refractory AML is still a substantial challenge and is associated with poor prognosis and low chance for cure, especially for elderly patients. Hence the necessity of new alternatives robust anti-leukemic activity while avoiding T-cell cytotoxicity against healthy tissues for treating AML patients. In this study, according to what has been published, we showed that the IL-1RAP protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared to the most common potential AML targets. After establishing the proof of concept for the efficacy of CART-cells targeting IL-1RAP in chronic myeloid leukemia (CML), we hypothesized that third-generation IL-1RAP CART-cells could eliminate AML LSCs. We first demonstrated that IL-1RAP CART-cells could be produced from AML T-cells at the time of diagnosis but also at relapse. Characterization of IL-1RAP CART-cells showed checkpoint markers expression at the end of the production process. In-vitro and in-vivo, we showed the effectiveness of IL-1RAP CART-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous CART-cells against primary cells from AML patients at diagnosis or at relapse. In patient-derived AML xenograft models (PDX), we confirmed that CART-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen and are cytotoxic against primary AML cells.
... Severe hypoxia, at 1% but not at 6% O2, down-regulated FLT3 expression in normal CD34 + hematopoietic progenitors in AML primary samples independently from the mutational state of FLT3 [100]. Under such conditions, the survival mechanisms of LSCs were based on phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling that it could be activated through signal transducer and activator of transcription 5 (STAT5)-mediated up-regulation of the tyrosine kinase receptor AXL [101]. ...
Acute myeloid leukemia (AML) is a hematologic malignancy caused by a wide range of alterations responsible for a high grade of heterogeneity among patients. Several studies have demonstrated that the hypoxic bone marrow microenvironment (BMM) plays a crucial role in AML pathogenesis and therapy response. This review article summarizes the current literature regarding the effects of the dynamic crosstalk between leukemic stem cells (LSCs) and hypoxic BMM. The interaction between LSCs and hypoxic BMM regulates fundamental cell fate decisions, including survival, self-renewal, and proliferation capacity as a consequence of genetic, transcriptional, and metabolic adaptation of LSCs mediated by hypoxia-inducible factors (HIFs). HIF-1α and some of their targets have been associated with poor prognosis in AML. It has been demonstrated that the hypoxic BMM creates a protective niche that mediates resistance to therapy. Therefore, we also highlight how hypoxia hallmarks might be targeted in the future to hit the leukemic population to improve AML patient outcomes.
... Hypoxic environments have been shown to promote central memory phenotype in CAR T-cells, which is beneficial for their functionality [156], but on the other hand reduce CAR T proliferation and hinder their effector memory differentiation and functionality (the latter being due to Tregs recruitment and enhancement of PD-L1 expression) [125]. Moreover, the hypoxic environment can alter the surface phenotype of AML cells in vitro [157], suggesting that the effect of hypoxia should be evaluated when selecting CAR T-cell targets to avoid antigen escape. A recent study illustrates how hypoxia-sensitive CAR T-cells, able to specifically activate in hypoxic sites (such as the AML-BM microenvironment) can be employed to avoid unwanted off-site toxicities [158]. ...
Simple Summary
Chimeric antigen receptor (CAR) T-cells are powerful therapeutic tools that have revolutionized the treatment of several hematological malignancies. However, their therapeutic application in acute myeloid leukemia (AML) remains challenging. In this review, the authors aimed to dissect how AML-leukemic stem cell and AML-bone marrow niche features can impact on the success of CAR T-cell therapy. The clinical implementation of some of the newly developed approaches discussed in this review may lead to the development of safe and effective CAR T-cell strategies for AML, accounting for the disease heterogeneity.
Abstract
Current treatment outcome for acute myeloid leukemia (AML) patients is unsatisfactory and characterized by high rates of relapse and poor overall survival. Increasing evidence points to a crucial role of leukemic stem cells (LSC) and the bone marrow (BM) leukemic niche, in which they reside, in AML evolution and chemoresistance. Thus, future strategies aiming at improving AML therapeutic protocols are likely to be directed against LSC and their niche. Chimeric antigen receptor (CAR) T-cells have been extremely successful in the treatment of relapsed/refractory acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma and comparable results in AML are highly desirable. At present, we are at the dawn of CAR T-cell application in AML, with several preclinical studies and few early phase clinical trials. However, the lack of leukemia-specific targets and the genetic and phenotypic heterogeneity of the disease combined with the leukemia-induced remodeling of the BM microenvironment are limiting CAR T-cell exploitation in AML. Here, we reviewed AML-LSC and AML-BM niche features in the context of their therapeutic targeting using CAR T-cells. We summarized recent progress in CAR T-cell application to the treatment of AML, and we discussed the remaining therapeutic challenges and promising novel strategies to overcome them.
... 47 Hypoxia was shown to down-regulate FLT3 and FLT3-ITD signaling in AML cells. 48 Under such conditions, the PI3K/AKT pathway was shown to sustain AML cell survival, rescuing FLT3 activity, 49 while AXL is known to activate PI3K/AKT signaling in AML cells. We and others have previously shown that cytokine-activated STAT5 enhances HIF2α expression directly 44 but also indirectly by inhibiting one of its destabilizers, the prolyl 4-hydroxylase domain protein 3 (PHD3) in both normal hematopoietic progenitors and leukemic cells. ...
... The previous studies have demonstrated that hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) can potentiate the anti-leukemia effects of cytarabine (Ara-C) [1,2]. Based on this rationale, CAG regimen [low-dose Ara-C, aclarubicin 1 3 are common in the adult M4/M5 AML patients, which are associated with poor prognosis [14,15]. Furthermore, M4/ M5 AML expresses significantly higher levels of GM-CSFR [8,16]. ...
High expression of granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor has been found in myelomonocytic or monocytic subtypes (M4/M5) of acute myeloid leukemia. Herein, we aimed to improve the effect of CAG [Ara-C, ACR, and G-CSF (granulocyte colony-stimulating factor)] regimen for acute monocytic leukemia by replacing G-CSF with GM-CSF. Results showed that the percentage of cells in S phase was higher with GM-CSF than with G-CSF treatment at 20 ng/mL (P < 0.05). When THP-1 and SHI-1 cells were primed with 20 ng/mL G-CSF or GM-CSF followed by Ara-C and ACR, cell proliferation rate in the CAGM (Ara-C, ACR, and GM-CSF) regimen was lower than in the CAG regimen (P < 0.05). Furthermore, CAGM regimen induced more obvious cell apoptosis than CAG regimen probably by reducing Bcl-2/Bax ratio (P < 0.05). Similar results were seen in primary cells from M5 patients. Collectively, our study suggests that priming with GM-CSF may be more effective than G-CSF in CAG regimen in acute monocytic leukemia.
... In addition, the hypoxic microenvironment downregulated FLT3 expression in AML cells, which was associated with suppression of the PI3K pathway. Reduced FLT3 expression led to a lack of response to cytarabine treatment [64]. ...
Abstract Drug resistance is of great concern in cancer treatment because most effective drugs are limited by the development of resistance following some periods of therapeutic administration. The tumor microenvironment (TME), which includes various types of cells and extracellular components, mediates tumor progression and affects treatment efficacy. TME-mediated drug resistance is associated with tumor cells and their pericellular matrix. Noninherent-adaptive drug resistance refers to a non-cell-autonomous mechanism in which the resistance lies in the treatment process rather than genetic or epigenetic changes, and this mechanism is closely related to the TME. A new concept is therefore proposed in which tumor cell resistance to targeted therapy may be due to non-cell-autonomous mechanisms. However, knowledge of non-cell-autonomous mechanisms of resistance to different treatments is not comprehensive. In this review, we outlined TME factors and molecular events involved in the regulation of non-cell-autonomous resistance of cancer, summarized how the TME contributes to non-cell-autonomous drug resistance in different types of antineoplastic treatment, and discussed the novel strategies to investigate and overcome the non-cell-autonomous mechanism of cancer non-cell-autonomous resistance.
