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The Question of Survival or Death: What Is the Role of Autophagy in Acute Myeloid Leukemia (AML)?

Autophagy plays a critical role in balancing sources of energy in response to harsh conditions and nutrient deprivation. Autophagy allows cells to survive in harsh condition and also serve as a death mechanism. Any dysregulation in autophagy signaling may lead to several disorders. Autophagy has been proposed to explain chemotherapy resistance in acute myeloid leukemia (AML). This signaling pathway can either act as a tumor suppressive function or chemo-resistance mechanism. Conventional chemotherapy drugs enhance apoptosis and indicate clinical benefit, but in some cases, relapse and chemotherapy resistance are observed. In leukemia, autophagy may promote cell survival in response to chemotherapy drugs. Therefore, new strategies by inhibiting or activating autophagy may find a broad application for treating leukemia and may significantly enhance clinical outcomes. In this review, we discussed how dimensional role of autophagy in leukemia.


INTRODUCTION
The word 'autophagy' means "self-eating". Autophagy consist of three forms including, microautophagy (act as a non-selective lysosomal degradative process) 1 , chaperone-mediated autophagy )selectively break down intracellular proteins in lysosomes) 2, 3 and macroautophagy (the most common form that cellular contents are degraded by lysosomes). Macroautophagy (herein refer to autophagy) is a process that cells from double-membrane structure, named autophagosomes which surround cellular contents and then fuse with lysosomes. Various stress conditions such as growth factor and nutrient deprivation 4,5 , endoplasmic reticulum stress, hypoxia, drugs and radiation are able to activate autophagy 6 . This process maintains cellular homeostasis by removing misfolded, damaged and ubiquitylated proteins 7,8 . Autophagy genes include 251 International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir Atg 1,3,4,5,7,8,9,10,12,16, ULK and beclin1. The ubiquitin-like proteins, including Atg8/LC3 and Atg12 have some structural similarity but do not have obvious homology with ubiquitin. Both proteins are induced by Atg7 and subsequently conjugated to phosphatidylethanolamine or Atg5, respectively. Beclin 1 is one member of a complex that involves the class III phosphatidylinositol (PtdIns)-3-kinase (also known as Vps34) that is stimulatory for the autophagy pathway. Class I PtdIns-3-kinases regulate the induction of the kinases mammalian target of rapamycin (mTOR) and Akt, which are known as an inhibitory for autophagy. Autophagy involved in different human diseases, including infectious diseases, neurodegenerative disorders and cancers. Autophagy has a role in both cell survival and programmed cell death 8,9 . The activity of autophagy in cancer treatment is a double edge sword. On the one hand, this pathway can be the driving force for the preservation of cancer cells. On the other hand, this signaling pathway act as an effective tumor suppressive, promote efficacious anti-cancer immunity and also keep normal cells from the toxicity of cancer treatments 5 . The complex function of autophagy in cancer continues to appear, and it is important for a scientist to clarify the mechanisms that how autophagy influences tumorigenesis and also therapy response 10 . Acute myeloid leukemia (AML) is defined by an accumulation of abnormal hematopoietic progenitor cells that exhibit the morphology of a certain phase of myeloid differentiation, in the peripheral blood and bone marrow [11][12][13] . Autophagy machinery has been proposed to play a critical role in hematopoietic malignancies such as AML [14][15][16] . The latest and conventional regimen (7+3 regimen) used for the treatment of AML that contain daunorubicin, idarubicin and cytarabine 17 . These chemotherapeutic agents have been found to induce autophagy in AML. Therefore, it is important to know the function of autophagy which induced through these agents. Nowadays scientists use the autophagy activator or autophagy inhibitors in combination with conventional medicine to promote the new strategy for cancer therapy. However, the function of autophagy during leukemia treatment is unknown 18 .

The autophagic Machinery
Autophagy is an early degradative pathway which happens in all eukaryotic cells . Molecular machinery of autophagy relies on recognition of ATG genes. Autophagy process can be divided into four stages: induction autophagy, the formation of autophagosome, degradation of the content within autophagosome and finally release of macromolecules from autophagolysosomes.

Initiation
Initiation of autophagy involves the group of ULK protein complex, including Atg13, ULK1,2, and FIP200 19 at the isolation membranes. This complex interacts with other ATG proteins to begin autophagosome formation. The kinase activity of ULK1 is controlled by the negative regulator mTOR complex 1 (mTORC1) during cellular stress 20 .

Nucleation
Autophagosome formation proceeds requires the organization of a large protein complex, known as Class III phosphatidylinositol-3-kinase (PI3K) complex

Elongation
Elongation of membranes requires Atg3, Atg4, Atg7, Atg10, and an Atg5-Atg12-Atg16L1 complex to conjugate phosphatidylethanolamine to the microtubule-associated protein1 light chain3 (LC3), cause translocation of LC3 from cytoplasm to the membrane of the forming autophagosomes. In addition acetylation and Phosphorylation of autophagy machinery proteins provide additional control over autophagosome formation 22 . After improvement of autophagosome formation, the Atg proteins are recycled in the cytosol except for LC3 which bound to the luminal membrane.

Maturation
LC3 related to luminal membrane remains associated in autophagosome and as soon as maturation into the autolysosome is degraded, while the LC3-II on the cytoplasmic surface can be recycled. Maturation of autolysosome characterized by fusion of autophagosomes with late endosomes which involves lysosomal organelles, endosomal multivesicular bodies and dissolution of the inner membrane 23 .  that bind to AKT. The kinase AKT is necessary for activating mTORC2 through GTPase-activating proteins, tuberous sclerosis complex 1&2 (TSC1&2) and Ras homolog enriched in brain (Rheb). Activation of this pathway has an inhibitory effect on autophagy. Growth factors induce the class I PI3K/Akt/mTOR signaling pathway which leads to the inhibition of autophagy. In the lack of growth factors, cells are incapable of taking up nutrients from the extracellular medium. The inhibition of mTOR in due to lack of growth factor consequently activate the autophagy machinery which leads to rescue cells from death via preserving ATP levels in starved cells 6 .

FOXO
Forkhead Box (FOX) O -characterized by a wingedhelix domain-transcription factors play a fundamental role in maintaining cellular redox homeostasis by activating genes involved in free radical scavenging and apoptosis. Provided a role for PI3K-Akt-mTOR signaling in regulating autophagy has been comprehended for some years, a role for FOXO transcription factors has only recently become clear. However, several studies have shown evidence that these transcription factors may act as fundamental regulators of autophagy 27 .

Bcl-2 family
Beclin 1(mammalian ortholog of yeast Atg6/Vps30) 28 is a haploinsufficient tumor suppressor gene which mostly monoallelically deleted in many human cancers including ovarian, breast and prostate. Beclin 1 cooperate with many autophagy proteins to initiate and maintain the autophagy pathway. This protein interacts with class ⅢPI3K to provide autophagosome formation 29 .in addition, Beclin 1 has been known as a direct association of Bcl-2 family 30 . The Bcl-2 family inhibits apoptosis through binding the proapoptotic effector proteins Bak, Bax and also BH3-only proteins 31 . The interplay between the autophagy protein (Beclin 1) and the antiapoptotic protein (Bcl-2) indicates a potentially main point of connection between apoptotic and autophagic machinery 21 . Scientist indicates the dual function of Bcl-2 family in autophagy regulation. Proapoptotic proteins such as BH3 proteins can enhance autophagy, whereas Bcl-2 family such as Bcl-2, Bcl-w and Bcl-XL as antiapoptotic proteins can prevent autophagy 21,32 .

ROS
Different levels of ROS can initiate signals in the various signaling pathway, such as autophagy. Many studies demonstrated that starvation can enhance the production of ROS and this could lead to autophagy induction.

P53
P53, which is known as a mutated gene in human cancers, also modulate mTOR activity. Nuclear P53 promotes translation of autophagy through activated AMPK, while cytosolic P53 inhibits autophagy by phosphorylation of AMPK 33 . The mitogen-activated protein kinase (MAPK) process regulates the maturation of autophagosomes. The role of AMPK in the maturation of autophagosome and lysosome fusion is not completely clear. AMPK can negatively regulate the mTORC1 pathway and mTORC1 activation can suppress maturation of autophagosome via UVRAG phosphorylation 34,35 .

Survival or death
Autophagy has been known as a necessary cellular process which requires for regulation of tumor progression and development. It also determines the sensitivity of cancer cells to anticancer therapy as well. However, the role of autophagy in tumor cells is complicated and depending on the conditions and have opposite roles in tumor proliferation. Both autophagic cell survival and autophagic cell death have been seen in many cancers 36 . Autophagy can be activated in tumor cells (the same as normal cells) through stress condition such as hypoxia and starvation 37 . This kind of environmental stresses combines with intrinsic metabolic stress which derived from the high metabolic requirement for metabolism and cell proliferation which lead to insufficient production of ATP. Autophagy is essential for tumor cells to survive metabolic stress. Genetic inhibition of autophagy inhibits survival in response to metabolic deprivation when apoptosis is inactivated 38,39 . Malignant cell activates autophagy in response to stress provide long-term survival, especially in defective apoptosis. As autophagy is a survival mechanism, it is possible that cause survival of established cancer cells in stress conditions 40 .
Tumor cells mostly encounter with defects in apoptosis which lead to permitting autophagy to keep survival by optimize nutrient utilization , metabolic stress or degradation of organelles 37,41 . In poorly vascularized tumors mostly cells encounter with hypoxia and metabolic stress lead to activation of autophagy signaling pathway 42 which can give an advantage to cancer cells to promote invasion and drug resistance. In addition to cell survival and cell proliferation, autophagy in some condition act as a programmed cell death (called programmed cell death type II 43 ) as well as in tumor suppression 44 . Different studies demonstrated that autophagic cell death is activated in tumor cells including colon 45 , breast 46 and prostate 47 in response to different anticancer drugs. In addition, autophagy defects have seen in different human tumors. Allelic loss of the required autophagy gene, beclin 1, is repeated in human ovarian, breast, and prostate cancer 37,48 . Furthermore, Loss of other autophagy regulators including Atg5 49,50 , Atg4C 51 and Bif-1 are also found 37 .

Autophagy inhibitor
Various research indicates that chemical inhibition or genetic knockdown of autophagy can significantly enhance cancer cell death which induced via antitumor medicine in preclinical models 10 5 . Rapamycin as an inhibitor blocks mTOR activity in vivo and also sensitizes tumors to apoptosis induced via chemotherapy 61 . Autophagy inhibitors and autophagy activators facilitate autophagy research and consequently facilitate its therapeutic potential in human diseases. As regards, most autophagy inhibitors are not quite specific subsequently genetic intervention can be a good choice to block autophagy, including RNAi and miR against Atg genes 57 .

AML
Approximately ten percent of all cancer mortalities result from leukemia and lymphomas in the Western world 62 . Based on improvements in lifestyle and health care, an average of lifespan is increasing whereas AML will be an increasing problem worldwide 62 . Acute leukemia is a highly heterogeneous class of malignant hematopoietic disorders which characterized by high proliferation of clonal neoplastic cells belonging to either the myeloid or lymphoid lineage 13,63 . AML is a heterogeneous disorder in both phenotypically and genetically. AML has been known as an aggressive disorder with high variability in clinical outcome. Different studies have been focused to determine mechanisms of leukemogenesis in AML. The LSC comes from cumulating of gene mutation including P53, NPM1 which effect on apoptosis and cell cycle regulation, AML1, CEBPA which effect on myeloid differentiation and FLT3, RAS, c-KIT, protein tyrosine standard phosphatase non-receptor 11 (PTPN11) which effect on cell proliferation 64 .
Mutations in upstream receptors such as FLT3, KIT and granulocyte colony-stimulating factor receptor have been found in AML which may induce activation of the JAk/STAT, Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling network that leads to inhibition of apoptosis 65 . In addition, upregulation of vascular endothelial growth factor receptor (VEGF-R) has been documented in AML, which can cause activation of these pathways 62 . Furthermore, overexpression of PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK frequently observed in AML patient 62 .

Autophagy and the hematopoietic niche
The main question is how stem cells adjust their selfrenewal and multipotency properties. Stem cells miss the potential for continued self-renewal when removed from their normal cellular environment, called stem cell niche, suggesting a vital role for the microenvironment in regulating stem cell behavior 66 . The two principal components of the hematopoietic niche are HSCs and mesenchymal stem cells. HSCs reside and self-renew in two distinct BM niches: the 'osteoblastic (endosteal)' and 'vascular' niches 67 . The BM niche adjusts HSC quiescence, differentiation, proliferation, and migration. HSCs interact with the niche through different molecular signals, including adhesion mechanisms 66,67 . The perfect adjustment of HSC quiescence and selfrenewal is not fully clear. LSCs Like normal HSCs associated with signals from the hematopoiesisregulating stromal environment for proliferation and survival. LSCs represent the unique characteristics as stem cells, including quiescence, pluripotency and self-renewal through the bone marrow microenvironment. However, the biology of LSCs shares lots of similarities with that of HSCs, LSCs are able to destroy HSCs, hijacking the bone marrow microenvironment. BM niches potentiate the growth of leukemia cells survival and drug resistance by providing the essential cytokines and cell contactmediated signals. On the other hand, LSCs themselves change the BM stromal cell composition or function that result in the survival of the leukemic cells 67 . Autophagy a general cellular housekeeping process is essential for differentiation and self-renewal of adult human stem cells, which mainly contribute to cytotoxic drug resistance, a long-known phenomenon of these cells but become sensitive against differentiation or blockage of autophagy 66 . Recent studies demonstrated that mechanisms of autophagy are active in HSC. For example, FIP200 is essential not only for the induction of autophagy but also for the maintenance role of HSC in vivo. Furthermore, HSC lacking ATG 7, another key autophagy protein, were also impotent to survive under in vivo situation 66 . Autophagy seems crucial for the equilibrium between quiescence, selfrenewal and differentiation of mesenchymal stem cells 68,69 . FOXO transcription factors play an essential role in HSC maintenance which has been proved in an original research by the conditional deletion of Foxo1, Foxo3a, and Foxo4 in the adult hematopoietic system resulting in lymphoid developmental abnormalities, myeloid lineage expansion, and a significant decrease of HSCs. at first, HCSs exhibited an effective long-term repopulating activity by downregulation of proteins involved in cell cycle by FOXO including cyclin G2, p130/Rb, p27, and p21 70 .
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir Figure 3.The bone marrow niche adjusts HSC quiescence, differentiation, proliferation, and migration. HSCs interact with the niche through different molecular signals. Leukemic blasts originate from common primary progenitor cells that have the capacity to pluripotency and selfrenewal through the bone marrow microenvironment. Conventional therapy for AML has been planned to eliminate leukaemic blasts. BM niches potentiate the growth of leukemia cells and wich consequently leads to drug resistance and metastatic of leukaemic cells via shielding LSCs.
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir

Role of autophagy in AML
Different studies indicate the double face of autophagy in cancer treatment . From one side, autophagy can promote maintenance of cancer cells, same as leukemia stem cells, or even play a role as a drug resistance mechanism enhancing cancer cell survival through self-digestion. On the other side, mechanism of autophagy can improve tumor suppressive by effective antitumor immunity while support healthy tissues from the toxicity of anticancer therapies 5 . some mutations in genes affecting autophagy, including beclin-1, PI3K, Akt, p53 and Bcl-2, have a role in the pathogenesis of malignant lymphoma, breast, prostate and ovarian cancer 71 . PI3K/Akt signaling pathway is activated in various cancers types and is a vital event in tumorigenesis 72 . Autophagy incriminates as the main mechanism through some antileukemic compound. Dysregulation of the PI3K/Akt/mTOR signaling pathway resulted in increasing chemotherapy resistance via inhibition of apoptosis, leukemic cell survival and also induce cap-dependent translation of mRNAs essential for differentiation, cell cycle progression and growth. initiation of this network is a common feature of leukemia and can dictate poor prognosis 73 . Therefore, it is reasonable to consider that targeting mTOR through rapamycin can effectively help to treat tumors. rapamycin derivatives potentially disrupt AKT activity in leukemic cells through 74,75 suppression of mTORC2 and mTORC1 pathway 76 . The fundamental action of the mTORC1 pathway has been discovered in many primary AML samples. In most AMLs cases, mTORC1 activation may be caused by an autocrine insulin-like growth factor-1 74 .
Alternative therapy for leukemia such as dasatinib 77 , arsenic trioxide 78 , vitamin D3 79 , histone deacetylase inhibitors 80 , and platonin inhibitor 81 induce autophagy as their mechanism of action. For instance, Histone deacetylase inhibitors can trigger apoptosis in myeloid leukemia blasts by suppressing autophagy, while platonin trigger autophagy programmed cell death in human leukemia cell lines. Chemotherapeutic drugs in the anti-AML treatment, induce autophagy including daunorubicin, idarubicin, cytarabine, vitamin D3 and Arsenic trioxide 11 . The induction of autophagy has recently been implicated in the regulation of leukemic cell death triggered by anticancer drugs 14 . As mentioned, autophagy can play as a tumor suppressor or cell survival in cancer cells. Hence, when autophagy acts as a cell survival, there is a potent rationale for the use of autophagy blockers in cancer treatment strategy 5 .
Anthracycline daunorubicin is one of the main antitumor agents broadly used in the treatment of myeloid leukemia. Unfortunately, the clinical efficacy of daunorubicin was limited because of its cardiac toxicity, renal toxicity and severe myelosuppression at the high dose chemotherapy. A group of the anticancer compound, such as DNAdamaging chemotherapeutic agents, have been indicated to increase the accumulation of autophagosomes in tumor cell lines. In a research, scientists indicated that daunorubicin induces autophagy via activation of MEK1/2 and ERK1/2 in myeloid leukemic cells. Inhibition of autophagy via autophagy inhibitors (chloroquine or Atg5/Atg7 knockdown) or inhibitors of MAPK increased the activation of caspases and tumor inhibition which induced by daunorubicin 82 .

Idarubicin
Idarubicin is an anthracycline antileukemic agent widely used for the treatment of AML, chronic myelogenous leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes. Scientists in an investigation reported that idarubicin induces autophagy in leukemia cell lines and primary leukemic cell through mTOR repression. Furtheremore they observed inhibition of Akt by Idarubicin in leukemic cells. Repression of mTOR activity by idarubicin may partly due to the blockage of Akt, since the Akt is one of the most leading activators of mTOR 83 .

Cytarabine
Cytarabine is another chemotherapeutic agent that used alone or in combination with other compound to treat different forms of leukemia. In a study, researchers indicated that cytarabine induces autophagy in leukemic cell lines through suppression of mTOR. In addition, they showed that inhibition of International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir autophagy by either pharmacological (Chloroquine) or genetic inhibitors enhance apoptosis in cytarabine-trated leukemic cells 84 .

Vitamin D3
Vitamin D3 plays vital roles in regulating physiological and cellular responses. Vitamin D3 potently inhibits cell proliferation in various cancer cells such as myeloid leukemia. It inhibits angiogenesis, tumor invasion, and metastases. Scientist demonstrated that vitamin D3 is able to induce autophagy though up-regulating beclin1 and they indicated that eliminating beclin1(beclin 1 act as an antiapoptotic role during vitamin D3-induced differentiation of the HL60 cell line) suppress autophagy and induce apoptosis 79 .

Arsenic trioxide
Arsenic trioxide has been reported to have strong antitumor effects in vitro and in vivo by increasing cell death, cell cycle arrest and apoptosis in leukemia cells. It has the main clinical activity in the treatment of patients with acute promyelocytic leukemia (a subtype of AML) 85 . In addition to apoptosis arsenic trioxide induce autophagy 86 through up-regulation of beclin1 87 . In addition, autophagy inhibitors have antiangiogenic features owing to their interference with endothelial cell survival 88 . As the role of bone marrow angiogenesis in human leukemia has been demonstrated 89 , hence another mechanism through which autophagy inhibitors can be beneficial is by affecting cancer cell vasculature 5 .

CONCLUSION
Autophagy is a self-degradative signaling pathway which has the main role in balancing of energy at crucial times in response to nutrient stress and development. Autophagy includes some sequential steps including, induction, insertion to lysosomes, degradation, and utilization of degradation products and each step may perform a different function 9 . Many studies of autophagy confirmed that this process of proteolysis has a function in the regulation of cell survival and death. Conventional regimen (daunorubicin, idarubicin and cytarabine) used for the treatment of AML which induced autophagy 17 . Hence, it is serious to know the function of autophagy which began with these agents. Recently scientists use the autophagy inducers or autophagy inhibitors in combination with conventional medicine to promote the new strategy for cancer remedy. However, the function of autophagy during leukemia treatment is not clear completely 18 . As findings discussed in this review, it is obvious that autophagy is affected by various therapeutic treatments used for AML. Autophagy functions are not always identical. Some when anti-cancer agents begin autophagy, whiles sometimes they prevent it. Sometimes this signaling pathway protects leukemic cells against cancer therapy, whereas sometimes it is essential for the remedy to be efficacious. Autophagy modulation, both stimulatory and repressive, can be attained by participating with different signaling pathways at multiple levels. The regulation of autophagy is complicated and includes many signaling pathways. Therefore, the safety and effectiveness of autophagy inhibitors and activators must be studied completely before clinical therapeutics development.