FLT3‐ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia

The FLT3‐ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins of the protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non‐mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.


| INTRODUCTION
Acute myeloid leukemia (AML) is the most frequent form of leukemia in adults and cause around 50% of leukemia related deaths. 1 The most frequent genetic alteration in newly diagnosed AML patients is the internal tandem duplication (ITD) of FMS-like receptor tyrosine kinase 3 (FLT3).This genetic event is present in nearly 30% of AML cases and characterize a fast-progressing disease with a poor patient outcome. 2,3Additionally, ITD mutations result in different peptide length and cellular localization. 4While the FLT3 wild-type protein is mainly localized in the cytoplasmic membrane, FLT3-ITD mutant forms are retained in the endoplasmic reticulum (ER) due to its impaired post-transcriptional and post-translational processing. 5The localization of the ITDs in the cell define differences in downstream effectors. 6,7Specifically, receptor localized in the cell membrane preferentially activate AKT and MEK, while ER retained receptors preferentially trigger STAT5 signaling. 8,9is is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
ER is responsible for multiple cell functions being the main cellular calcium storage and so regulating calcium homeostasis and signaling 10 and also the major site of lipid biosynthesis. 11Furthermore, ER regulates the synthesis, folding, maturation and posttranslational modifications of secreted and transmembrane proteins. 12Several perturbations can disrupt ER homeostasis resulting in the accumulation of misfolded proteins, a condition called "ER stress".
In case of stress, ER triggers the Unfolded Protein Response (UPR) that aims to reestablish protein homeostasis. 13Importantly, UPR plays a major role in leukemogenesis and the use of UPR modulating drugs as monotherapy or in combination with standard of care has emerged as a new therapeutic strategy for AML. 14though it is well-known that ITD of the FLT3 gene result in the retention of a constitutively active form of this receptor in the ER and the subsequent modification of its downstream effectors, 8 the role that its accumulation in the ER plays in the functionality of this cell organelle remains unknown.Here we show that, unlike wild-type FLT3 AML cells, accumulation of FLT3-ITD in the ER results in changes in protein processing, as well as in calcium homeostasis, preventing calcium transfer to mitochondria that affects the glycolytic metabolism of AML cells and provides a survival advantage to tumors carrying these ITD mutations.
For experimental procedures, cells between passage 3-20 were seeded at a density of 5 � 10 4 cel/mL.LookOut® mycoplasma qPCR detection kit (Sigma Chemical Co.) to was used to ensure mycoplasma-free cultures.Cell culture reagents were from Sigma (Sigma Chemical Co.), except for FBS, which was purchased from GIBCO (Invitrogen Life Technologies, Barcelona, Spain).Culture flasks and dishes were acquired from Thermo Fisher Scientific.All other reagents were purchased from Sigma (Sigma Chemical Co.), unless otherwise indicated.

| Evaluation of cell viability
Cells were seeded in 6-well plates.After treatments, cells were harvested and resuspended in 400 μL of PBS and 100 μL of 0.4% (w/ v) trypan blue solution which uptake is indicative of irreversible membrane damage preceding cell death.The number of cells (proliferation) and the percentage of viable and non-viable cells (viability) were determined using an automatic cell counter (Coun-tess™ 3, Invitrogen Life Technologies, Spain).ex.512 nm/em.581 nm; Rodamine 123: ex.488 nm/em.515 nm; DCFH-DA: ex.485 nm/em.530 nm; ThiolTracker Violet: ex.400 nm/ em.520 nm).The obtained fluorescence was normalized with total number of cells determined using an automatic cell counter (Countess™ 3, Invitrogen Life Technologies, Spain).

| Evaluation of LDH activity
Cells were seeded in 24-well plates and determination of LDH activity was accomplished following specifications of the lactic dehydrogenase based In Vitro Toxicology Assay Kit (Sigma-Aldrich).
Absorbance was determined using an automatic microplate reader (μQuant; Bio-Tek Instruments, Inc.) at 490 nm and then the obtained data were relativized with total protein concentration.

| Western Blot
For protein expression analysis, cells were lysed in ice-cold lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 2 μg/mL aprotinin, 1 μg/mL pepstatin-A, 110 nM NaF, 1 mM PMSF, 20 mM Tris-HCl pH 7.5).Thirty to 50 μg of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Amersham Bioscience, Pittsburgh, PA, USA).Blots were incubated overnight at 4°C with appropriate antibodies (Supplemental Table S1).Immunoreactive polypeptides were visualized using horseradish peroxidase conjugated secondary antibodies (anti-rabbit or anti-mousse IgG peroxidase conjugated 1:4000; Santa Cruz Biotechnology) and enhanced-chemiluminescence detection reagents (Merck Millipore) following manufacturer-supplied protocols.Experiments were repeated at least three times, and data were calculated as the average � Standard Error.Significance was tested by t-test when two groups were compared, while one-way ANOVA followed by a Student-Newman-Keuls multiple range test was used to multiple range test.Statistical significance was accepted when p ≤ 0.05.

| FLT3-ITD plays a role in protein folding machinery in AML cells
FLT3-ITD is known to be abnormally retained in the ER due to its defective glycosylation.Since abnormal protein accumulation in the ER normally triggers UPR, we therefore evaluated expression of three ER resident proteins controlling UPR response, IRE1α, PERK and ATF6, in FLT3-ITD mutant and wild-type AML cells.Unexpectedly, we found that FLT3-ITD mutant AML cells present lower levels of the active forms of these UPR effectors compared to wild-type cells (Figure 1A) as well as lower levels of two ER stress marker proteins KDEL and BiP (Figure 1B), suggesting lower UPR activation in mutant cells.
Differences in UPR activation are known to dictate cell response to ER stress inducers.We therefore treated AML cells with the ER stress inducers DTT and β-mercaptoethanol affecting protein folding.
These compounds preferentially induced cell death in wild-type cells, having a minimum effect in FLT3-ITD mutant cells (Figure 1C,D).On the contrary, treatment with thapsigargin, that disrupt calcium homeostasis in the ER, resulted in the induction of cell death regardless of FLT3 mutational status (Figure 1E).These data suggests that AML cells carrying FLT3-ITD are more resistant protein folding inhibitors.
In line with notion, protein folding in the ER is an oxidative process that relies mainly on Endoplasmic Reticulum Oxidase 1 (ERO1α) and Protein Disulfide Isomerase (PDI). 16 ERO1α and PDI levels reveled that FLT3-ITD mutant cells have a higher expression of at least one of them compared to wild-type cells (Figure 2A), being ERO1α overexpressed in all mutant cells.Moreover, similar results were found in an isogenic model.Murine FLT3-ITD expressing cells (Ba/F3 ITD cells) presented higher expression of ERO1α and increased activation of STAT5 -main downstream effector of ER retained FLT3-ITD-compared to Ba/F3 wild-type cells (Figure 2B).Additionally, inhibition of FLT3 signaling with midostaurin -an FDA-approved FLT3 kinase inhibitor-in human cell lines led to a specific decrease in the expression of ERO1α only in mutant cells (Figure 2C), further suggesting that mutant receptor regulates ERO1α expression in AML cells.
ERO1α oxidoreductase activity during protein folding is known to led to H 2 O 2 generation. 17Evaluation of intracellular peroxides revealed that both human ITD cells and Ba/F3 ITD cells present lower H 2 O 2 levels compared to wild-type cells (Figure 2D).Moreover, ITD cells (both human cells lines and Ba/F3 ITD cells) also present reduced levels of glutathione (Figure 2E), which is consumed on each protein folding cycle and can be also responsible for detoxification of the newly formed peroxides.All these data suggest a more efficient protein folding activity in FLT3-ITD cells.induction of cell death while no effect was found in wild-type cells (Figure 3A).These data suggest that ERO1α is essential for the survival of FLT3-ITD mutant cells.In line with these data, EN460 increased the efficacy of cytarabine -the main chemotherapeutic drug in AML treatment-(Figure 3B) as well as of midostaurine (Figure 3C), suggesting a role of this oxidoreductase in the chemoresistance of ITD AML cells.

| ERO1α regulates glucose metabolism in FLT3- ITD mutant AML cells
In addition to participate in the protein folding process, ERO1α also plays an important role in the regulation of other cellular functions.
In fact, ERO1α can be also located in other intracellular compartments outside ER, 22 mainly on the mitochondrial associated membranes (MAM)-interaction points between the ER and mitochondria-wild-type regulating Ca 2þ flux from ER to mitochondria through the protein complex formed by the IP3R-GRP75-VDAC proteins. 23Immunoblotting reveals that despite having higher level of ERO1α expression, FLT3-ITD cells present lower levels of IP3R-GRP75-VDAC proteins, suggesting the presence of fewer points of interaction between ER and mitochondria (Figure 4A).Isogenic Ba/F3 also reveals that ITD expressing cells present lower levels of GRP75 and VDAC (Figure 4B).Interesting, ERO1α inhibition using EN460 increases the expression of IP3R-GRP75-VDAC proteins in mutant cells (Figure 4C), suggesting that ERO1α can not only be located at MAM, but it can also play a key role in their formation.Ca 2þ flux from ER to mitochondria at MAM is essential for the proper function of the enzymes involved in the Krebs cycle and therefore the regulation of the glycolytic metabolism of cells.Thus, evaluation of mitochondrial calcium in AML cells reveals that wild-type cells present higher calcium levels compared to ITD cells (Figure 4D), which correlated with the increased expression of IP3R-GRP75-VDAC protein complex.These increased levels of mitochondrial calcium also correlated with an increase in mitochondrial membrane potential (Figure 4E), which reflects the activity of the electron transport chain.Similar results were found in the isogenic Ba/F3 model with a decrease in mitochondrial calcium and mitochondrial membrane potential in ITD expressing cells in comparison to wild-type expressing cells (Figure 4F).According to this wild-type cells also display lower levels of LDH activity compared to mutant cells (Figure 4G).Similar results were found in FLT3-ITD expressing Ba/F3 cells which presented both increased levels of glucose uptake and LDH activity (Figure 4H).These data reinforce the idea that wildtype AML cells harbor a preferentially mitochondrial metabolism, while mutant cells seem to be mostly glycolytic.Accordingly, inhibition of glycolytic metabolism by using oxamate and 3-bromopiruvate induces cell death only in ITD cells (Figure 5A), while inhibition of mitochondrial metabolism with oligomycin and rotenone resulted in the opposite effect (Figure 5A).
Involvement of IP3R-GRP75-VDAC protein complex in the stimulation of mitochondrial metabolism in FLT3 wild-type cells was confirmed by treatment with the GRP75 inhibitor MKT-077.GRP75 inhibition resulted in an increase in glucose uptake, LDH activity and a decrease in mitochondrial membrane potential (Figure 5B) suggesting a shift from mitochondrial glucose metabolism to glycolytic metabolism.
We have shown that IP3R-GRP75-VDAC complex levels anticorrelate to those of ERO1α.Moreover, our data also suggest that FLT3 mutational status dictates the route for glucose metabolism, and it does so likely in an ERO1α depending on manner.According to this concept, treatment of AML FLT3-ITD mutant cells with the ERO1α inhibitor EN460 resulted in a drop in glucose uptake and in LDH activity, as well as an increase in mitochondrial calcium uptake and in mitochondrial membrane potential (Figure 5C).This finding is significant because the FLT3-ITD mutation is associated with a poor prognosis in AML.
Tumor microenvironment is characterized by inadequate vascularity, limited nutrient and oxygen availability. 25These, along with the high demand for protein synthesis due to rapid tumor cell proliferation, result in greater ER stress and UPR activation compared to normal cells.While this phenomenon has been extensively observed in solid tumors, emerging evidence indicates the involvement of the UPR in hematological tumors. 26In contrast to solid tumors, hematological tumors are less exposed to external factors inducing ER stress.Therefore, intrinsic factors, such as the presence of mutated proteins, are more likely to cause ER stress in these cells.A prevalent example is the FLT3-ITD mutation, which occurs in approximately 30% of AML cases.The mutant protein accumulates in the ER due to incorrect processing within this cellular compartment.Surprisingly, despite the accumulation of improperly processed proteins typically causing ER stress and UPR activation, our data indicate that the presence of the FLT3-ITD mutation reduces ER stress and UPR.In these sense, scarce expression of UPR-related proteins in AML patients with the FLT3-ITD mutation has been previously described. 27vertheless, these findings underscore the complexity of the UPR and its regulation in different tumor types, including AML.Understanding how specific mutations impact the UPR could potentially unveil novel therapeutic strategies.
Lower ER stress and UPR activation in FLT3-ITD mutant cells is related to more efficient mechanism of protein folding due to an increased expression of ERO1α.The use of EN460, a pharmacological inhibitor of ERO1α, revealed that increased expression of this protein in mutant cells is crucial for their survival and contributes to the cells' resistance to conventional chemotherapeutic drugs.In fact, antitumor effect of ERO1α inhibition has already been described previously in hematological tumors. 28This finding suggests that targeting the mechanism by which FLT3-ITD regulates ERO1α expression may offer a potential strategy to enhance the efficacy of current chemotherapies and improve outcomes for AML patients with the FLT3-ITD mutation.
In addition to its role in protein folding, ERO1α is also enriched in MAMs facilitating calcium flux towards the mitochondria. 23Surprisingly, our findings show that FLT3-ITD cells, despite exhibiting higher levels of ERO1α expression, have lower intramitochondrial calcium.
However, the localization of ERO1α in MAMs is not solely determined by its increased expression.Instead, it is influenced by factors such as the oxidative state of the ER and the availability of oxygen. 29 the same way, MAM conformation is influenced by other cellular processes, such as UPR.In line with this, our assessment of MAM proteins showed a noticeable increase in the expression of the IP3R-GRP75-VDAC complex in wild-type cells, which is associated with the higher UPR levels observed.This complex is primarily responsible for facilitating calcium transfer to the mitochondria, which plays a crucial role in Krebs cycle enzyme activity and mitochondrial glycolytic metabolism. 30Interestingly, our research highlights differences in glucose metabolism based on the FLT3 mutational status, with FLT3-ITD cells favoring glycolytic metabolism and showing lower levels of IP3R-GRP75-VDAC proteins.GRP75 inhibition wild-type resulted in a shift from mitochondrial glucose metabolism to glycolytic metabolism in AML wild type cells, mirroring the changes observed in mutant cells.These findings align with previous studies, suggesting that the FLT3-ITD receptor promotes a Warburg-type metabolism in AML cells. 31,32Understanding the metabolic changes driven by FLT3-ITD and its impact on calcium flux and ERO1α expression may offer valuable insights into potential therapeutic strategies for AML.Inhibition of ERO1α in mutant cells demonstrated an anticorrelation between the expression of ERO1α and the expression of the MAM proteins which significantly influenced glucose metabolism.While ERO1α 0 s role in facilitating calcium transfer to the mitochondria and its impact on mitochondrial metabolism have been wellestablished, a recent study has highlighted its connection with glycolytic metabolism in pancreatic cancer.Zhang et al. conducted research on pancreatic cancer patients' samples and found a positive correlation between ERO1α expression and the mRNA levels associated with glycolytic metabolism.Remarkably, when ERO1α was silenced, glucose uptake and lactate production decreased, suggesting a reduced reliance on glycolysis. 20 summary, our study provides valuable insights into the molecular mechanisms underlying the influence of FLT3-ITD mutation on ER homeostasis in AML cells.Thus, mutated receptor regulates the expression of ERO1α contributing to the homeostatic state of the ER decreasing UPR.This is not only going to be key in the survival and resistance to the treatments of the cells, but also is going to be decisive for the type of metabolism that these cells present, being the mitochondrial pathway the predominant one in those with a higher UPR (wild-type cells) and the glycolytic pathway the predominant one in those with lower UPR (mutated cells).Further research in this area could lead to the development of targeted therapies aimed at disrupting the ER folding mechanism in mutant cells, potentially improving treatment outcomes for AML patients with this mutation.
Evaluation of F I G U R E 1 FLT3 mutational status regulates ER stress response in AML cells.(A), protein expression of P-IRE, P-PERK and s-ATF6 in wildtype and ITD mutant AML cells.GAPDH expression has been used as loading control.*p ≤ 0.05 versus mutant cells.(B), protein expression of UPR markers KDEL and Bip in wild-type and ITD mutant AML cells.GAPDH expression was used as loading control.*p ≤ 0.05 versus mutant cells.(C-E), cell death after 48 h treatment with thapsigargin 10 nM (C), DTT 1 mM (D) and β-mercaptoethanol 1 mM (E) in wild-type and ITD cells.*p ≤ 0.05 versus untreated cells.

4. 2 |
ERO1α plays an essential role in FLT3-ITD survival and chemoresistance Even though high ERO1α expression has been associated to poor clinical outcome in several type of cancer, 18-21 little is known about its role in AML.According to the overexpression of ERO1α, treatment of mutant AML cells with the ERO1α inhibitor EN460 resulted in the F I G U R E 2 Mutated FLT3 receptor regulates protein folding machinery.(A), protein expression of ERO1α and PDI in wild-type and ITD mutant AML cells.GAPDH expression has been used as loading control.*p ≤ 0.05 versus mutated cells.(B), protein expression of ERO1α and P-STAT5 in Ba/F3 cells expressing wild-type or FLT3-ITD receptor.Actin expression has been used as loading control.Dashed line represents levels of expression in Ba/F3 cells expressing wild-type receptor.*p ≤ 0.05 versus wild-type expressing cells.(C), protein expression of ERO1α AML cells after 24 h treatment with 500 nM midostaurin in OCI-AML3 (wild-type) and MV-4-11 (mutant) cells.GAPDH expression has been used as loading control.Dashed line represents levels of untreated cells.*p ≤ 0.05 versus untreated cells.(D), intracellular peroxide levels in wild-type and mutant AML cells (both human cell lines and murine Ba/F3 cells).*p ≤ 0.05 versus mutant cells.(E), GSH levels in wild-type and mutant AML cells (both human cell lines and murine Ba/F3 cells).*p ≤ 0.05 versus mutant cells.
These data indicate that ERO1α inhibition in AML mutant cells switches their metabolism from glycolytic to mitochondria.This altered metabolic state induced by ERO1α inhibition creates a potential weakness in AML cells. 24F I G U R E 3 ERO1α participates in survival and chemoresistance of mutant cells.(A), cell death after 48 h treatment with 10 μM EN460 in wild type and ITD mutant AML cells.*p ≤ 0.05 versus untreated cells.(B), cell death after 48 h treatment with cytarabine (2 µM) and EN460 (5 µM for MV-4-11 and 2.5 µM for MOLM-13) alone or in combination in mutant cells.*p ≤ 0.05 versus untreated cells.#p ≤ 0.05 versus individual treatment.(C), cell death after 48 h treatment with midostaurin (500 nM for MV-4-11 and 200 nM for MOLM-13) and EN460 (5 µM for MV-4-11 and 2.5 µM for MOLM-13) alone or in combination in mutant cells.*p ≤ 0.05 versus untreated cells.#p ≤ 0.05 versus individual treatment.TUROS-CABAL ET AL.F I G U R E 4 ERO1α favors glycolytic metabolism in ITD AML cells.(A), protein expression of P-IP3R (active form), GRP75 and VDAC in wild type and ITD mutant AML cells.GAPDH expression has been used as loading control.(B), protein expression of P-IP3R (active form), GRP75 and VDAC in wild type Ba/F3 cells and ITD Ba/F3 cells.Actin expression has been used as loading control.*p ≤ 0.05 versus Ba/F3 cells expressing wild-type receptor.(C), protein expression of P-IP3R, GRP75 and VDAC in mutant cells after 24 h treatment with 10 μM EN460.Actin expression has been used as loading control.Dashed line represents levels of untreated cells.*p ≤ 0.05 versus untreated cells.(D), mitochondrial calcium levels in wild-type and mutant AML cells.*p ≤ 0.05 versus mutant cells.(E), mitochondrial membrane potential (∆Ѱ) in wild-type and mutant AML cells.*p ≤ 0.05 versus mutant cells.(F), mitochondrial calcium levels and mitochondrial membrane potential (∆Ѱ) in wild type Ba/F3 cells and ITD Ba/F3 cells.*p ≤ 0.05 versus Ba/F3 cells expressing wild type receptor.(E), LDH activity in wild-type and mutant AML cells.*p ≤ 0.05 versus mutant cells.(F), glucose uptake and LDH activity in Ba/F3 cells expressing wild-type or FLT3-ITD receptor.*p ≤ 0.05 versus Ba/F3 cells expressing wild type receptor.Our results indicate that the mutational status of the FLT3 receptor plays a crucial role in influencing ER homeostasis in AML cells.Cells carrying the mutated FLT3 receptor show lower levels of ER stress and reduced activation of the UPR compared to non-mutated cells.