Lysosomes in Stem Cell Quiescence: A Potential Therapeutic Target in Acute Myeloid Leukemia.

Lysosomes are cellular organelles that regulate essential biological processes such as cellular homeostasis, development, and aging. They are primarily connected to the degradation/recycling of cellular macromolecules and participate in cellular trafficking, nutritional signaling, energy metabolism, and immune regulation. Therefore, lysosomes connect cellular metabolism and signaling pathways. Lysosome's involvement in the critical biological processes has rekindled clinical interest towards this organelle for treating various diseases, including cancer. Recent research advancements have demonstrated that lysosomes also regulate the maintenance and hemostasis of hematopoietic stem cells (HSCs), which play a critical role in the progression of acute myeloid leukemia (AML) and other types of cancer. Lysosomes regulate both HSCs' metabolic networks and identity transition. AML is a lethal type of blood cancer with a poor prognosis that is particularly associated with aging. Although the genetic landscape of AML has been extensively described, only a few targeted therapies have been produced, warranting the need for further research. This review summarizes the functions and importance of targeting lysosomes in AML, while highlighting the significance of lysosomes in HSCs maintenance.

1. Introduction

In 1955, Christian de Duve discovered lysosomes in hepatic tissue while investigating the cellular localization of glucose-6-phosphatase hydrolase, an enzyme thought to be connected with the mechanism of insulin action. He found that glucose-6-phosphatase hydrolase was located in “sac-like particles” that store nutrients, metabolites, and waste materials in a cell [1]. This breakthrough discovery led to the identification of several other additional extracellular and intracellular macromolecule-digesting hydrolases that were pH sensitive, such as phosphatases, nucleases, glycosidases, proteases, peptidases, sulfatases, and lipases. Collectively, these enzymes are capable of hydrolyzing almost all classes of macromolecules.

Lysosome research gained major attention after the discovery of autophagy, where lysosomes are involved in a degradative cellular process required for recycling waste materials and maintaining cellular homeostasis. However, in autophagy, understanding of lysosomes was limited to them as cellular waste disposal compartments [2,3]. More recently, lysosomes have been identified as stress sensors and the coordinator of cellular responses to various environmental stimuli, including nutrients, growth hormones, and immunological signals [3,4]. These multifaceted roles of lysosomes have produced clinical interest in this organelle as a potential therapeutic target for various biological disorders, including cancer [5]. This review summarizes the involvement of lysosomes in the pathogenesis of malignant illness, including blood cancer, and in the maintenance of hematopoietic stem cells (HSCs).

2. Biological Properties and Functions of Lysosomes

Lysosomes vary in shape, location, and function depending on the species and cell types [6]. Interestingly, lysosomes rapidly alter their distribution, amount, size, and activity to meet various cellular requirements. The outer lysosomal membrane is densely packed with transmembrane proteins, notably lysosome-associated membrane proteins (LAMPs), including LAMP1, LAMP2, LAMP3, LAMP4, and LAMP5. LAMP1 and LAMP2 are the most abundant lysosomal membrane proteins, contributing up to ~80% of all lysosomal membrane proteins. Other vital lysosomal membrane proteins (LMPs) include ion channels and a variety of cargo receptors, such as the Niemann–Pick C1 protein (NPC1), synaptotagmin (SYT7), chloride channel protein 7 (CLC7), and vacuolar (V)-ATPase proton (H+) pump (V-ATPase) [7]. At the expense of ATP hydrolysis, the V-ATPase pump transports H+ against its concentration gradient to maintain a lysosome pH between 4.5 and 5.5, which is required for the degradation of macromolecules by luminal hydrolases [8].

Endosomes and Golgi-derived vesicles containing lysosomal-specific hydrolytic enzymes are utilized to synthesize primary lysosomes [9]. The digestion of cargo is a vital function of lysosomal enzymes. The fusion of lysosomes is thought to be the primary mechanism by which internal or external cargo is degraded, and resulting metabolites are exported back to the cytoplasm for metabolic reuse or cell growth [10]. Lysosomes serve as metabolic signaling hubs and degradative compartments, influencing cell fate. They receive cargo from various routes, including the autophagic, endocytic, and phagocytic pathways. It is well known that lysosomes have multiple roles as degradative, clearing, and nutrition reservoirs [11]. Lipids, carbohydrates, proteins, nucleic acids, and damaged mitochondria are engulfed in lysosomes, where they are degraded by enzymes in the acidic lysosomal environment. The degraded products are recycled and reused in metabolic processes, stored in the lysosomal lumen for later use, or secreted by exocytosis [9,12].

The lysosomal luminal compartment acts as a hydrolytic engine, producing acidic hydrolytic enzymes such as proteases, nucleases, lipases, glycosidase phosphatases, and sulfatases. The decreased activity of these enzymes has the potential to impair cellular homeostasis. Lysosomes are vital to cellular health due to the storage of essential ions and metabolites, such as calcium, iron, and zinc, as well as hydrogen (H+), sodium, potassium (H+/Na+/K+), chloride (Cl−), and adenosine triphosphate (ATP). The amino acid (AA) levels, such as arginine and leucine, are regulated by lysosomal receptors involved in external signaling [13].

3. Subcellular Localization of Lysosomes

The subcellular location of lysosomes inside the cell determines their signaling characteristics. Kinesin motors of the kinesin superfamily (KIF) proteins (KIF1Bβ and KIF 2A) and a small GTP binding protein, ADP-ribosylation-factor-like GTPase 8B (ARL8B), allow lysosomes to migrate from the center of cells to the periphery and vice versa [14]. Environmental cues such as nutrition signaling also impact the lysosomal localization. Under nutrient-deprived conditions, lysosomes’ peripheral localization near the cell membrane suppresses mTORC1 activity and induces autophagy, whereas their perinuclear localization induces autophagy and increases interaction with mTORC1 [15].

Mechanistically, the FYVE-domain proteins protrudin and FYVE and coiled-coil domain autophagy adaptor 1 (FYCO1) help to guide mTOR-positive lysosomes to move towards the plasma membrane in a VPS34-dependent process [16]. Under serum starvation, perinuclear clustering of lysosomes inhibits the reactivation of mTORC2 [17]; however, under hypoxic conditions, lysosomes become dispersed, and mTORC1 activation is inhibited [18]. A tumor suppressor protein, folliculin (FLCN), controls the lysosome interaction with the perinuclear membrane, thus limiting lysosome location [19]. Importantly, lysosomal subcellular location determines the pH of the lysosomes. The acidity of the lysosomal membrane is reduced when V-ATPase activity is inhibited [20]. Lysosomal exocytosis, in which lysosomes fuse with the plasma membrane, requires a Ca2+ regulated process of lysosome mobility within a cell that is also engaged in various physiological activities such as plasma membrane repair and immunogenic ATP release [21].

4. Lysosomes as a Signaling Hub

Lysosomes, like other organelles, interact, communicate, and signal primarily at their surface to connect external signals to the cellular metabolism networks. Numerous ion channels and signaling proteins are present on the membrane of lysosomes.

Growth factors and energy status

mTOR complex 1 (mTORC1), which is a vital signaling mechanism, fuels anabolic/biosynthetic pathways and inhibits catabolic processes such as autophagy. mTORC1 is regulated by a range of factors, such as energy status, growth factors, and nutrition. Tuberous sclerosis complex (TSC) is a signal complex that is composed of TSC1, TSC2, and TBC1D7 subunits. TSC negatively regulates the activity of mTORC1. TSC2 acts as a GTPase-activating protein (GAP) for Rheb and inactivates it by keeping it in a GDP-bound state. TSC2 inactivation results from either insulin or insulin-like growth factor (IGF) signaling. Activated Rheb, a protein in a GTP-bound state, activates mTORC1 [22]. Energy deficit cellular state induces the activation of AMP kinase at the lysosomal surface, which activates TSC2 via phosphorylation and inhibits RAPTOR activity, leading to the inhibition of mTORC1 and stimulation of catabolic pathways [22].

Cytosolic amino acid (AA) signaling

Cytosolic AA promotes mTORC1 translocation to the lysosome surface (Figure 1), which itself is mediated by the coordinated actions of many complexes, including the Ragulator and Rag GTPases (A, B, C, and D). The Ragulator complex comprises five subunits: Lamtor1/p18, Lamtor2/p14, Lamtor3/MP1, Lamtor4/p10, and Lamtor5/HBXIP. The Lamtor/p18 subunit is anchored to the lysosomal membrane in response to AA signaling. Ragulator complex plays a role of guanine nucleotide exchange factor for Rag A/B and promotes their GTP-bound state. This translocates both the RAG GTPases and mTORC1 to the lysosomal membrane, where mTORC1 gets activated by the small GTPase Rheb [23,24].

Figure 1

The lysosome surface is a hub of signaling activity. Several proteins found in lysosomal membranes are involved in various signaling cascades. The vacuolar-type H+ ATPase (V-ATPase) is a proton pump that regulates pH. mTORC1 connects metabolism and signaling. Intra- and extra-luminal amino acid signaling may affect the mTORC1 signaling pathway’s components. The amino acid transporter SLC38A9 detects arginine in the lysosomal lumen and activates mTORC1 via the Rag GTPases and Ragulator complex. A leucine sensor in the cytosol, Sestrin 2, controls mTORC1 activity with the assistance of GATOR proteins. mTORC1 also affects lysosome biogenesis adversely. Ca2+ triggers the initiation of lysosome biogenesis. Once Ca2+ is released into the cytoplasm, it dephosphorylates TFEB, allowing it to translocate to the nucleus, where it aids in the transcription of the CLEAR network genes and activates lysosomal and autophagic transcription. Transmembrane proteins such as LAMPs are involved in autophagy, lipid transport, and immunological response. SYT7 is a calcium-dependent membrane protein involved in lysosomal exocytosis.

Additionally, the presence of specific AA residues modulates the activity of additional GEFs and GTPase-activating proteins (GAPs), which are downstream of GTP-RagA and GDP-RagC signaling and mTORC1 signaling. The GTPase-activating proteins toward Rags (GATOR) complex is a critical integrator of amino acid signaling [25]. GATOR has two sub-assemblies, namely GATOR1 and GATOR2. GATOR1 functions as an off switch for RagA/B by releasing mTORC1 from the lysosome, thus functioning as a negative regulator. GATOR2 positively regulates mTORC1 activity by inhibiting the GAP activity of GATOR1. Leucine and arginine residues act through stress-inducible proteins (Sestrins) and cytosolic arginine sensors for mTORC1 subunit 1 (CASTOR1). On amino acids binding, Sestrin and CASTOR1 dissociate from GATOR2, releasing their suppressive effects on GATOR2 and activating mTORC1 [26]. If AA is unavailable, mTORC1 activity can be regulated by cyclin-CDK inhibitor 1C (CDKN1B) and/or p27, which are tumor suppressors and bind to LAMTOR1, preventing mTORC1 activation and thus activating macroautophagy [27].

A transporter protein, SLC38A9 facilitates the efflux of AA such as leucine from the lysosome lumen [13,28,29]. Increased intraluminal leucine levels also activate mTORC1 via ATP hydrolysis by the V-ATPase, which promotes the recruitment of mTORC1 by the Ragulator/Rag complex [30] (Figure 1). SLC38A9 in the presence of AA interacts with the Rag GTPase-Ragulator complex, which activates mTORC1 [28,29,31,32]. The tumor suppressor FLCN-FNIP complex activates RagC/D through its GAP activity. FLCN-FNIP complex, which acts as a GAP for Rag C/D, translocates to the lysosomal surface in the absence of AA [33,34]. Together, the Ragulator and FLCN/FNIP activities convert the Rag complex into its active form, which is recognized by the raptor subunit of mTORC1 [35], triggering its translocation to the lysosomal surfaces [36,37,38].

Taken together, these results suggest that lysosomes not only make a significant contribution to the cellular catabolism by providing nutrients for cell development but also serve as a platform for nutrient sensing and metabolic signal processing.

5. Regulation of Anabolic/Catabolic Pathways and mTROC1

The mTORC1 fuels anabolic/biosynthetic pathways while inhibiting catabolic processes such as autophagy (Figure 1). mTORC1 and transcription MiT/TFE transcription factor phosphorylation regulate anabolic/catabolic pathways by regulating mTORC1. The protein TFEB, which is phosphorylated on two sites in the cytoplasm, is translocated to the cytoplasm under nutrients. During stress, such as starvation, mTORC1 inhibitor induces TFEB migration to the nucleus, which initiates CLEAR network gene transcription [3]. Calcineurin, a Ca2+-dependent phosphatase, is another regulator of TFEB nuclear translocation. TRPML1/mucolipin-1 releases Ca2+ under starvation and triggers calcineurin, which dephosphorylates TFEB. Sixty or more genes, including microtubule-associated protein 1 light chain 3 b (Map1lc3b) and WD-repeat domain and phosphoinositide interacting 2 (Wipi2), which are involved in lysosome biogenesis and autophagy, were shown to be inhibited by a zinc finger family DNA-binding protein (ZKSCAN3). Furthermore, ZKSCAN3 and TFEB are inversely influenced by starvation [39]. The identification of a stress-induced lysosome-to-nucleus signaling mechanism through TFEB validates the importance of lysosomes in cellular signaling.

6. Lysosomal Intra-Luminal Compartment Signaling Events

The lysosomal luminal compartment acts as a hydrolytic engine that stores acidic hydrolytic enzymes. Any imbalance in the activity of these enzymes may have a significant effect on cellular homeostasis. Besides enzymes, lysosome also store various important ions and metabolites such as Ca2+/Fe2+/Zn2+, H+/Na+/K+, Cl−, and ATP. Additionally, lysosomal receptors control the intra-luminal levels of amino acids such as arginine and leucine and are implicated in external signaling [13].

Lysosomes and calcium signaling

The lysosomal membrane consists of numerous ion channels that aid in establishing concentration gradients and maintaining the lysosome membrane potential [40]. Three distinct types of Ca2+ channels in mammalian lysosomes are reported: transient receptor potential mucolipin subfamily (TRPML)/mucolipin 1-3, two-Pore (TPC1-2), and P2X purinoceptor 4 (P2 × 4). Additionally, they respond to various cues, including cell stress, ATP depletion, phospholipids, and nutrition [41]. Lysosomes act as mobile intracellular Ca2+ storage, with 5000-fold higher concentrations of Ca2+ than the cytosol of the cell [42]. They uptake Ca2+ from the cytosol in a pH-dependent manner. Ca2+/H+ exchanger promotes lysosomal Ca2+ uptake, and recent investigations have discovered that the endoplasmic reticulum (ERs) Ca2+ levels may serve as an independent source for lysosome Ca2+ reserves [43,44,45].

Lysosomes contact and fuse with other organelles such as endosomes to create hybrid organelles in which the bulk of the endocytosed cargo is degraded. Various GTPases and SNARE complexes regulate them, and Ca2+ is released from the lysosome lumen [46]. Many research groups have reported that lysosomal processes (including mobility, trafficking, and creation of membrane contact sites) are regulated by lysosomal Ca2+ release [47,48,49,50]. The exchange of Ca2+ between the two organelles is accomplished through the ER-lysosome membrane contact sites, which are facilitated by inositol 1,4,5-trisphosphate receptors (IP3Rs), with a potential Ca2+ uptake channel/transporter in the ER/lysosome membrane [42,51]. Ca2+ concentrations in the lysosome are elevated compared to the cytoplasm, which is a primary regulator of various lysosomal functions.

These results imply that the lysosome is an essential regulatory center for several pathways involved in cell proliferation and differentiation.

Essential amino acid sensing function of lysosomes

The lysosome can recognize both luminal and cytosolic amino acid (AA) levels, with the crosstalk between these pathways maintaining homeostasis or responding to nutrient-related signals. The sodium-coupled amino acid transporter SLC38A9 detects lysosomal luminal arginine. SLC38A9 undergoes a conformational change in response to arginine, which stimulates the Rag A/B (Ras-related GTP-binding) GTPase and Ragulator complex on the lysosomal surface. This activates the mechanistic target of rapamycin complex 1 (mTORC1), which is one of two protein kinase complexes incorporating the serine-threonine kinase mTOR. The mTORC1 complex consists of mTOR kinase, Raptor, GL, and DEPTOR proteins. Furthermore, mTORC1 is activated by Rheb (Ras homolog abundant in the brain) GTPase on the lysosomal membrane [52,53]. Simultaneously, SLC38A9 facilitates the efflux of other AA into the cytosol, such as leucine [13,28,29]. Increased intraluminal leucine levels also activate mTORC1 via ATP hydrolysis by the V-ATPase, which promotes the recruitment of mTORC1 by the Ragulator/Rag complex [30] (Figure 1). Taken together, these results suggest that lysosomes not only make a significant contribution to cellular catabolism, which provides nutrients for cell development, but also serve as a platform for nutrient sensing and metabolic signal processing.

Lysosomal Cell Death

Lysosomal-dependent cell death (LCD) is a kind of controlled cell death that uses intra-lysosomal components such as cathepsins or iron translocation caused by lysosomal membrane permeabilization (LMP) to enhance or initiates apoptosis, autophagy, proptosis, and ferroptosis (Figure 2).

Figure 2

Impairment of the lysosome induces apoptosis. Apoptosis is triggered by lysosomal damage in a mitochondrial-dependent manner. BID is cleaved into tBid by damaged lysosomes, which increases BAX oligomorphism via inducing Bcl-2 degradation by cathepsins. As a result, BAX is translocated to the mitochondrial outer membrane (MOM), where it interacts with mitochondrial permeability y transition pore (MPTP) to release cytochrome C (Cyto C) and triggers apoptosis. Necroptosis occurs when lysosomal activity is inhibited, resulting in the accumulation of necrosome components (such as RIPK1 and RIPK3) and the release of hydrolyzed caspase 8. The necroptosis executor (MLKL) is phosphorylated and translocated to the cell membrane or organelle membrane, resulting in necrosis. Pyroptosis is induced by damaged lysosomes through cleavage of GSDMD into GSDMD-N by releasing CTSG, activation of NLRP3, and caspase-1 by the release of CTSB. Pyroptosis is induced by damaged lysosomes through the cleavage of GSDMD into GSDMD-N by the release of cathepsin G and the activation following that. Pyroptosis results in cell perforation and release of large amounts of interleukin-1 and interleukin-18.

Cathepsins (CTS), a group of lysosomal proteases that are classified as serine (CTSA and CTSG), aspartic (CTSD and E), or cysteine proteases (CTS B, C, F, H, K L, O, S, V, W, and X). Several research studies have demonstrated that most lysosomal enzymes are stable and active in a neutral and acidic pH environment and retain their degradation potential [54,55,56]. CTSB is the most stable protease at physiological pH and induces apoptosis [57,58,59,60]. CTSD is involved in apoptosis induced by interferon-g, Fas/CD95/APO-1, TNF-a, oxidative stress, and sphingosine [61]. CTSL is the least stable lysosomal protease at neutral pH and is a crucial regulator of UV-induced keratinocyte death [62,63,64]. The most abundant lysosomal proteases are CTSB, CTSD, and CTSL, which are found in most tissues [65].

Beyond protein breakdown, CTS has several other cellular functions [66], including cancer progression [56,67]. Extracellular CTS have been associated with matrix disintegration, cell migration, and cancer cell invasion [68]. Interestingly, CTS released from the lysosomal membrane induces cell death [69] and exhibits necrotic, apoptotic, or apoptosis-like features (Figure 2) [70]. Although much remains unknown, it has been shown that lysosomes play a critical role in the resistance to and initiation of cell death and the final clearance stage of cell death.

Lysosomal regulation of immune responses

Lysosomes are involved in several steps of immune responses, including pathogen detection, phagocytosis, antigen processing, and inflammation. The sentinel cells, such as macrophages and dendritic cells (DCs), use toll-like receptors (TLRs) to recognize pathogen-associated molecular patterns [40]. Members of the TLR family, including TLR 3, TLR7, TLR 8, and TLR9, are located on endolysosomes. TLR9 senses mitochondrial DNA, which is transported to the lysosome for mitophagy, a process of removing damaged mitochondria [71,72]. When phagosomes mature, they fuse with lysosomes to form phagolysosomes, degrade foreign materials, or damaged organelles. A transcriptional factor, TFEB, increases phagocytosis in a calcium-dependent mechanism that activates immune-related genes [73]. Macrophages, DCs, and B cells are antigen-presenting cells (APCs) that engulf pathogens and display processed antigens on the major histocompatibility complex (MHCs) at their surfaces [74]. Lysosomal pH is critical for antigen processing because a highly acidic environment with low pH in the lysosomal lumen causes excessive proteolysis of the engulfed microorganism and reduces cross-presentation. On the other hand, increased lysosomal pH may affect lysosomal degradation potential and impede antigen presentation, as found in lupus disease [75]. In addition to their role in antigen processing, lysosomes also regulate the levels of several pro-inflammatory cytokines, such as IL-1β and IL-18, which are selectively degraded via autophagy (Figure 2) [76,77].

These results reveal that lysosomes play an essential role in modulating the intensity of the immune response and in regulating inflammation.

7. Galectins

Studies have shown that galectins mediate lysophagy, a process of removing damaged lysosomes [78]. Galectins play a vital role in inflammation, immunological responses, cell migration, autophagy, and signaling. Fifteen mammalian galectins (Gal-1–15) have been discovered to date [79,80]. It has been shown that apoptosis, cell cycle regulation, and nuclear splicing of pre-mRNA are all regulated by intracellular Gal-3. Many biological processes are affected by Gal-3′s interactions with various intra- and extracellular proteins under physiological and pathological conditions, such as development, immunological responses, and cancer [80]. Importantly, galectins have also been linked to maintaining leukemic cells in the tumor microenvironment and AML prognosis [81]. Extensive investigation of the lysosomal galectin may reveal their essential roles in a wide variety of biological processes, including AML, HSC biology, and other diseases.

8. Lysosomes and Autophagy

Autophagy is a process by which cells recycle their excess or damaged organelles using lysosomes, and degraded products are recycled back to the cytosol for further use. Autophagy helps cells adapt to extreme environmental conditions, such as nutritional deficiency, removing harmful/damaged organelles, destroying pathogens, and removing protein aggregates [82,83]. Autophagy is mediated by three pathways: (i) nucleotide, (ii) chaperone, and (iii) nucleotide with chaperone-dependent activities. The cargo is processed via multiple protein conjugation steps and is transported from the autophagosome to the lysosome for their degradation [84]. The ATG (autophagy-related) genes that are important for the autophagy process were discovered in yeast and subsequently found in other species [85]. The mammalian macroautophagy process begins with the formation of two major complexes: (I) the class III phosphatidylinositol 3-kinase (PI3K-III/VPS34) complex containing Beclin-1 (a mammalian homolog of yeast Atg6), hVps34, p150 (a mammalian homolog of yeast Vps15), and Atg14-like protein (Atg14L), and (II) the ULK1 kinase complex (ULK1-Atg13-FIP200-ATG101)—this activates the class III PI3K/Beclin-1 complex, enabling autophagosome nucleation formation (Figure 3). Both the above complexes are regulated by phosphorylation in the following pathways:

Figure 3

Autophagy and lysosomal pathway. Cytoplasmic cargo is delivered to lysosomes through autophagy. Macroautophagy includes compartmentalizing cytoplasmic payloads in LC3-coated double-membrane vesicles, known as autophagosomes. Structures known as autolysosomes are formed when autophagosomes fuse with lysosomes, and lysosomal hydrolases break down the damaged organelle. The autophagic lysosome reformation process recycles lysosomal components from autolysosomes once cargo breakdown is complete. The recognition of cytoplasmic substrates containing an accessible KFERQ-like motif by HSC70 is vital for chaperone-mediated autophagy. The HSC70-substrate complex is recognized at the lysosomal membrane by LAMP2A. LAMP2A oligomerization then forms a translocation channel, transporting the substrate into the lysosomal lumen. The LAMP2A channel disassembles into monomers after substrate delivery, allowing substrate delivery. Microautophagy entails the transport of cargo into the lysosomal lumen via interaction. Direct interaction into the endosomal lumen during endosomal microautophagy transport cargo to late endosomes, where it forms multivesicular bodies. The lysosomes then join the multivesicular bodies to degrade the cargo. Endosomal microautophagy delivers cargo via ESCRT-machinery.

The PI3K/AKT/mTOR signaling pathway: In the abundance of nutrients and growth factors, the PI3K/AKT/mTOR signaling pathway is activated, while autophagy is suppressed and vice versa.

AMP-activated protein kinase (AMPK): AMPK is a crucial energy sensor that monitors the ratio of ATP/AMP or ATP/ADP. Under nutrients starvation, condition-activated AMPK phosphorylates TSC2 and inactivates it, which further induces autophagy mediated by ULK-1/2 complex formation [86].

Chaperone-mediated autophagy (CMA): CMA is a client-specific, selective autophagic pathway (Figure 3). CMA clients contain a signature motif containing a short stretch of 5 AA residues, namely KFERQ (lysine, phenylalanine, glutamate, arginine, glutamine) [87]. The HSP70 family of proteins—such as chaperone protein HSPA8, also known as HSC70 or HSP73—recognizes KFERQ [88]. A single molecule of LAMP-2A binds to the client protein on the lysosomal surface. Lysosomal HSP90 stabilizes multimers of LAMP-2A receptors on the lysosomal surface, facilitating the transfer of the client. Substrates are transported to the lysosome through the HSPA8-LAMP-2A interaction [89].

Microautophagy is mediated by lysosome membrane dynamics, allowing cytosolic contents to be engulfed and scavenged into the lumen for degradation. Microautophagy molecular mechanisms are less understood than the other two pathways. Like CMA, the protein clients have a KFERQ-like motif and are transported to the lysosomes by HSPA8 via the endosomal sorting complex needed for the transport (ESCRT) mechanism [90]. The ESCRT machinery (Figure 3) helps protein migrate into multivesicular bodies (MVBs), which are a unique form of the late endosome, that can then fuse with the lysosome [91].

9. Role of Lysosomes in Maintaining Stem Cell Quiescence

Quiescence is required for maintaining a stem cell pool. Adult stem cells are dormant but are capable of exiting dormancy and quickly expanding and differentiating in response to stress. The quiescent state of stem cells is required for their self-renewal and is a critical factor for determining cancer stem cells’ (CSCs) susceptibility to chemotherapy and targeted treatments [92,93]. Thus, molecular underpinnings of adult stem cell quiescence are crucial for targeting quiescent CSCs in various malignancies. Recent research has increased the knowledge of the intrinsic and extrinsic regulatory mechanisms that regulate stem cell quiescence.

In the mouse brain, lysosome- and proteosome-associated gene expression was high in quiescent and active neural stem cells (NSCs), which is critical for neurogenesis [94].

NSCs:

Quiescent NSCs (qNSCs) have a higher number of and bigger lysosomes, demonstrating compromised activity compared to active stem cells. Lysosomal gene expression is enhanced with quiescence, while their degradative capacity is reduced. These bigger lysosomes store more protein aggregates, and lysosomal activity is compromised, leading to a weak response of qNSCs to stress. On the other hand, enhancing lysosome activity reduced protein aggregates with the activation of qNSC [94]. Age-dependent reduction in the lysosome numbers with more protein aggregates inhibits qNSCs activation [94].

Interestingly, in old qNSCs, increased activity of TFEB in response to growth factors restored activation, suggesting that enhanced lysosomal function promotes activation of old NSCs. In addition, protein aggregation in lysosomes is associated with aging, which can be ameliorated by activating qNSCs [94]. On the contrary, a higher in vitro lysosome protease activity was reported in qNSC in vitro, with high TFEB activation and reduced NSCs growth in the adult mouse brain [95]. Lysosome function during quiescence and neurogenesis may differ depending on the cellular niche and NSCs’ age [96].

Mitf gene family members Tfe3 and Tfeb are essential for regulating lysosome biogenesis in quiescent rat embryonic fibroblasts [97]. The lysosome-based signaling system is a driver of mouse embryonic stem cell differentiation [98]. Interestingly, disrupting the distribution of lysosomal enzymes promotes the nuclear translocation of TFE3 and enhances the self-renewal of mouse embryonic stem cells (ESCs) [98]. MYC has an antagonistic impact on TFE3 in neoplastic cells and human iPSCs [99]. AMPK null embryonic stem cells suppress mTORC1 activation in the lysosome, and phosphorylation of TFE shows severe differentiation abnormalities. Due to TFEB hypophosphorylation and decreased nuclear localization, AMPK−/− ESCs retain pluripotency but fail to produce chimeric embryos. Embryoid body development requires TFEB and appropriate lysosome activity for endodermal differentiation, which it undergoes through regulation of canonical Wnt signaling [100]. Recent research has shown that regulating autophagy is a potential strategy for enhancing the biological characteristics of mesenchymal stem cells (MSCs) [101]. These results indicate that lysosome biogenesis control is essential for stem cell self-renewal or neoplastic cell self-renewal.

HSCs:

Like qNSCs, quiescent hematopoietic stem cells (qHSCs) express more lysosomal genes and contain expanded lysosomes (Figure 4A) [102]. HSCs self-renew and remains quiescent/dormant to contribute to the expansion of the stem cells pool. When qHSCs get activated, it differentiates to produce different blood cells. qHSCs reside in a hypoxic niche of bone marrow and have modest metabolic needs, which are supplied by glycolysis [103,104]. A recent study revealed that human HSCs displayed reduced mitochondrial activity under stead-state conditions, signifying the importance of low mitochondrial activity for HSC maintenance [105]. HSCs actively maintain low nutrient sensitivity to maintain their quiescent non-dividing state. HSCs store and utilize nutrients to generate energy to adjust their activity, although the precise mechanism remains unclear. There has also been disagreement with the long-held belief that glycolysis is the primary energy source in quiescent HSCs (Figure 4B) [102]. The glycolytic pathway was more closely connected with active than quiescent HSCs under normal homeostasis, and inhibiting glycolysis in vivo enhances the potency/ quiescence of activated HSCs [102]. These results suggest that glycolysis is an energy source for cycling cells, which is true for embryonic stem cells and cancer cells [103]. Lysosomes were found to be critical in the regulation of HSC metabolism. Lysosomal degradation suppressed glycolysis and oxidative phosphorylation in the activated HSCs [102]. Lysosomes appear to play a critical role in maintaining the equilibrium between HSC quiescence and activation [102,106]. It has been shown that highly purified phenotypically defined HSCs have large lysosomes [102]. Lysosomes numbers were high in qHSCs, but few are inactivated/primed HSCs (Figure 4A,B). In addition, qHSCs had a lower lysosomal degradative potential than activated HSCs [102].

Figure 4

Lysosomes are abundant in quiescent HSCs. (A) Schematic representation shows quiescent HSCs are abundant in lysosomes. (B) Schematic representation shows quiescent HSCs are abundant in lysosomes and have poor mitochondrial lysosomal clearance. HSCs are primed by acidification and activation of lysosomes, possibly through mTORC1 activation. Lysosomes keep HSCs dormant by sequestering and storing old and defective organelles and proteins; lysosomal breakdown and release of metabolites coincide with and contribute to HSC activation and priming. (C) Lysosomes are critical for the maintenance of HSCs due to their asymmetric inheritance, which affects cell fate during cell division.

Liang et al. further showed that suppressing lysosomal activity using V-ATPase inhibitor restores activated HSCs to qHSCs (Figure 4B), while also suppressing lysosomal activity, restoring slow lysosomal degradative potential, enhancing the potency of activated HSCs in vivo, and inhibiting mTORC1 signaling. These results suggest that lysosomal function in qHSCs is critical to their stem cell capacity [102]. Moreover, it is known that inhibiting lysosomal function decreases autophagy, which decreases the potency of HSCs [103,104].

Autophagy relies on the lysosome-mediated self-degradation process and is crucial for HSCs homeostasis, especially for old HSC [107]. Lysosomes and autophagy modulate stem cell fate [95,102,108]. HSCs with minimal replication potential divide asymmetrically, resulting in two different types of daughter cells. The one with highly functional lysosomes demonstrates higher regenerative potential than the other with less functional lysosomes and more differentiation capacity (Figure 4C) [108,109,110].

Different stem cell types exhibit varying degrees of dormancy, and quiescence is governed by common and exclusive pathways. According to a recent finding, TFEB activates the endo-lysosomal pathway and promotes quiescence by reducing HSCs’ metabolic, mitogenic activity. Furthermore, increased MYC inhibits lysosomal degradation and activates HSCs [111]. Lysosomes control the pace of degradation and output of molecules such as amino acids required for HSC quiescence [102]. Galectins have also been linked to the regulation of HSCs differentiation and self-renewal [112] and AML prognosis [81]. Jia et al. showed that Gal-3 is required for qHSC maintenance. Nuclear factor kappa B (NF-κB)-mediated AKT activation led to enhanced Gal-3 production, which prevented cell-cycle progression and activated HSCs [113].

Therefore, alteration of lysosomal activity may be used to augment the potency of dormant human HSCs in transplantation settings, to combat cancer stem cells, or to improve stem cell function more broadly, including old neural stem cells, and possibly old HSCs.

10. Autophagy in HSCs

Autophagy is critical for stem cell activity and hematological homeostasis in neonates, demonstrating a new feature of autophagy involvement in HSC regulation [114]. Macroautophagy is critical to HSCs’ energy metabolism. The transcription factor FOXO3A activates a gene expression pathway that promotes autophagy and protects HSCs from stress [115]. Atg7 deletion in the hematopoietic system of mice caused severe clinical symptoms such as lethargy, piloerection, and weight loss, leading to the death of the animals within 12 weeks; this was associated with the increased number of mitochondria and DNA damage [116,117,118]. Atg5 knockout mice also showed similar pre-leukemic characteristics [119]. The absence of focal adhesion kinase family interacting protein of 200 kD (FIP200), a ULK-interacting protein that is required for autophagosome nucleation process, is implicated in the maintenance of fetal HSCs, and its absence causes a defect in erythroid maturation, decrease in HSCs’ reconstituting capacity, and abnormal increase in numbers of myeloid cells [120]. Previous reports have shown that ATG5, ATG7, and ATG12 are required to maintain a mature HSCs pool [107,116,121]. Recently CMA has been shown to be critical for regulating protein quality and metabolic reprogramming during HSCs activation, where pharmacological activation of CMA restored the functionality of old mice and human HSCs [122]. ATG6 autophagy-related 6 homologs (also known as Becn1) is the first identified mammalian autophagy gene that participates in the phagophore nucleation [123]. Becn1 deficiency disrupted blood system homeostasis and reduced HSCs’ reconstitution efficiency [124].

Mitophagy is a type of autophagy that removes damaged mitochondria and regulates HSCs homeostasis. Ito et al. showed that silencing of Pink1, a putative kinase that regulates mitophagy, or Parkin, which encodes an E3 ubiquitin ligase and is required for Pink1 binding to the mitochondrial outer membrane, induces mitophagy and impairs in vitro expansion of HSCs [125]. Atad3a, a regulator of Pink1-dependent mitophagy, impaired hematopoietic lineage commitment and increased hematopoietic stem and progenitor cell (HSPC) pools in a conditional Atad3a knock-out mice [126]. The macrophage-erythroblast attacher (MAEA), a membrane-associated E3 ubiquitin ligase component, is required for HSC maintenance by enhancing autophagy [127].

11. Lysosomes in Cancer

A variety of normal cellular functions depend on lysosomes and lysosomal enzymes. Lysosomes are essential in increasing cellular biomass production and the ability of a cell to adapt to nutritional stress, which is also required for cancer development and cellular transformation. A cancer cell contains more active lysosomes than a healthy cell [128], and they demonstrate high levels of mTORC1 signaling, catabolic reactions, and autophagy, which aid in cancer cell survival, metabolism, and proliferation [129]. In addition, Machado et al. reported that a decrease in lysosomal neuraminidase 1 (NEU1) enhances lysosomal exocytosis and lysosomal hydrolase activity, which remodels the extracellular matrix within the tumor to invade neighboring tissue, which promotes cancer metastasis [130]. Chemotherapeutic drugs get sequestered in the lysosomes, and therefore lysosomes also have an essential role in inducing chemoresistance [131]. Furthermore, metastatic cells are more vulnerable to lysosome-targeting drugs because lysosomes of metastatic cells are highly diverse in size, content, location, and activity compared to normal cells [132].

Several lysosome-dependent biological processes are attractive targets for cancer treatment (Figure 5). Multiple therapeutic drugs are designed to target different lysosomal functions to treat various disorders such as cancer. Lysosomal targeting drugs increase the lysosome’s luminal pH, which inactivates CTHs or destroys the lysosomal membrane, promoting LCD [133]. A summary of the various lysosomal targets is shown in Table S1.

Figure 5

Different strategies for modulation of lysosomes and their functions. Lysosomes are known to link with many signaling pathways, allowing for multiple targeting options. Since autophagic pathways play different roles in cancer, various methods exist to inhibit/suppress or activate lysosomes. Inhibiting mTORC1 may indirectly enhance autophagy. Lysosomotropic chemicals have the potential to disrupt the membrane and thus prevent lysosome fusion. Inhibitors of V-ATPase have an impact on several pH-sensitive lysosomal enzymes. HSP70 inhibitors may be used to enhance the impact of lysosomotropic drugs that protect against HSP70. Lysosomal exocytosis releases cathepsins into the extracellular environment, promoting extracellular matrix breakdown and malignant cell invasion. Cathepsin protease inhibitors have the potential to be utilized as a treatment option.

12. Lysosomes in Acute Myeloid Leukemia (AML)

AML is a hematological condition in which an abnormally high number of immature blood-forming cells accumulate in the bone marrow, impairing the formation of normal blood cells. Despite advances in the pathogenesis and treatment of AML, AML-associated relapses remain a challenge. Lysosomal mTORC1 regulates cellular metabolism, differentiation, proliferation, and cell death [134], and lysosomal autophagic pathways are also necessary for hematopoiesis. Lysosomes contribute to the development of various hallmarks of blood cancers, such as sustained proliferative signaling (mTORC1 signaling), metabolism (catabolic reactions, autophagy), and invasion (lysosomal exocytosis) [135]. TFEB, a transcription factor that regulates autophagy and lysosome formation, is also known to be an oncogene that drives renal cell carcinoma [136,137]. Several common chromosomal deletions observed in AML are found in autophagy genes involved in cell death and differentiation [119]. Autophagy inhibition promotes the survival and proliferation of AML cells [119]. Yun et al. showed that MYC-regulated TFEB expression is dynamically controlled during myelopoiesis. TFEB acts as a tumor suppressor that induces normal and malignant myeloid progenitor cell differentiation and cell death. Lysosomal disruption affects AML progenitor cells, encouraging lysosomal targeting in AML [138].

Auer rods are lysosome-derived crystalline cytoplasmic inclusion bodies found in acute promyelocytic leukemia (APL), AML, and myelodysplastic syndromes. Auer rods contain several lysosomal enzymes and display different shapes such as needles, commas, diamonds, rectangles, or corkscrews [139]. The average number of Auer rods per cell varies in AML patients and could be important for the diagnosis of myeloid cell neoplasms, but they do not have any prognostic value [140]. The role of lysosomal tabulation/fusion in Auer rods synthesis and their exact role in AML are unknown. However, Auer rods have been linked to the activation of APCs in cancer. The activation of macrophages and DCs induces significant tubulation of endo-lysosomes, which improves Auer rods’ surface area to volume ratio and transport of MHC-II peptides to the cell surface [141].

13. Autophagy in AML

AML is one of the most common types of leukemia among older adults and is characterized by a cessation of myeloid differentiation leading to uncontrolled proliferation and prolonged life span of cells [142]. AML cells display a lower level of autophagy than non-leukemic or differentiated cells, and defects in autophagy can trigger or exacerbate AML [119,143]. Autophagy blockade results in the accumulation of autophagy cargo receptor SQSMT1/p62 and has been reported in acute promyelocytic leukemia (APL) cells during neutrophil development [144]. APL is characterized by an accumulation of immature blood-forming cells (promyelocytes) in the blood and bone marrow due to the depletion of white, red, and platelet cells.

Furthermore, it has been found that microRNAs, such as miR–17, –20, –93, and –106, all of which downregulate SQSMT1/p62, are more abundant in mouse and human blast cells than in a differentiated blood cell, neutrophil [144]. SQSMT1/p62 is believed to prevent the accumulation of ubiquitinated proteins [145]. SQSMT1/p62 is required for cell proliferation, and mitochondrial integrity and mutations in the SQSMT1/p62 affect mitophagy and myeloid leukemia development [146].

HSCs with heterozygous deletion of Atg5 in a mixed-lineage leukemia–eleven nineteen leukemia (MLL-ENL) mice model showed a high proliferation rate and severe leukemia features with a metabolic shift to glycolysis, an important hallmark for cancer development [119], and also enhanced leukemia features in another MLL-AF9 mouse model [147]. On the other hand, inhibition of autophagy followed by Atg5 or Atg7 deletion reduced leukemia-initiating cells (LICs) and prolonged the lifespan of leukemic mice. LICs are a rare subset of leukemic cells with stem cell features. Atg7-depleted LICs exhibited high mitochondrial activity and more reactive oxygen species (ROS) production. Additionally, Atg7 deletion in LICs induced apoptosis, which significantly reduced the number of peripheral blood leukemic cells, indicating a greater reliance of peripheral blood leukemic cells on autophagy for their viability [148].

Numerous studies support the notion that the role of autophagy in leukemia development varies depending on the oncogene that can affect the progression of the disease [149]. Receptor tyrosine kinase (RET) is a proto-oncogene that has recently been shown to be a critical kinase in the progression and development of AML [150]. Interestingly, RET-activated pathways reduce autophagy and stabilize leukemogenic drivers such as mutant FLT3. Furthermore, inhibition of RET leads to FLT3 depletion via autophagy. Interestingly, proteasome inhibitors promote FLT3-ITD degradation through autophagy [150]. Inhibition of FLT3-ITD mutant in AML cells, on the other hand, affects autophagy-dependent proliferation both in vitro and in vivo, indicating that FLT3-ITD promotes a high level of basal autophagy. The expression of the ATF4 transcription factor is required for FLT3-ITD-dependent autophagy [151]. In AML cells with mutant nucleophosmin 1 (NPM1) protein, promyelocytic leukemia (PML) protein remained and stabilized in the cytoplasm. PML’s cytoplasmic location induces phosphorylation of AKT, which then stimulates a pro-survival autophagy process [152].

In addition to ATG genes, H2.0-like homeobox transcription factor (HLX) is overexpressed in AML and promotes AML cell survival via AMP-activated protein kinase (AMPK)-induced autophagy levels [153]. Autophagy in AML also promotes fatty acid oxidation through lipophagy, which is required for mitochondrial oxidative phosphorylation (OxPHOS), a defining feature of chemotherapy-resistant cells. Nonetheless, autophagy’s role in AML chemoresistance is contextual and could be either cytoprotective or cytotoxic depending on the drugs used [149]. Overall, the role of autophagy in AML pathogenesis is diverse, and it could be either a tumor promoter or tumor suppressor.

14. Targeting Lysosomes in AML

Lysosome is a multifunctional organelle and a therapeutic target in various types of cancer, including AML. Further, lysosomes are larger in AML cells than normal cells, rendering AML cells more vulnerable to lysosome-targeting chemicals [154]. Thus, blocking the synthesis of lysosomal membrane protein (LMPs) may be utilized to induce apoptosis in AML cells while preserving normal hematopoietic stem cells and preventing the development of chemotherapy resistance. As a result, disrupting the autophagy–lysosome function effectively delays the onset and progression of malignancies, and it is progressively emerging as a potential target for tumor therapy. Lysosomes can be targeted in AML using various strategies (Figure 6).

Figure 6

Large lysosomes contribute to resistance to chemotherapy in cancer. (A) In cancer cells (AML), enlarged lysosomes may make them more resistant to chemotherapy by concealing therapeutic chemicals inside lysosomes. (B) Lysosome-targeting therapies in cancer (AML): The lysosome membranes are damaged, enabling enzymes to release into the cytosol. Disruption of lysosomal function due to hyper- or hypo-acidification increases medication resistance. Inhibiting pump (v-ATPase) or channel activity may induce lysosome-dependent cell death.

Interfering with lysosomal luminal homeostasis and direct lysosomal structural damage

Targeting lysosomes is one of many approaches to target chemoresistance in cancer. Lysosomotropic agents are chemicals that accumulate in the lysosomal lumen and enhance the lysosomal pH, leading to lysosomal dysfunction and LMPs [69]. Commonly used in vitro lysosomotropic drugs include chloroquine, ammonium chloride, methylamine, and siramesine [155,156] (Table S1). Lysosomotropic agents prevent the sequestration of drugs in the lysosomal lumen, making them available in the cytosol and reaching their target. Combining two lysosomotropic agents, vincristine and siramesine, showed better anti-tumor activity than either treatment alone in breast cancer [156,157]. Mefloquine, a lysosome-damaging agent, releases lysosomal CTSB and L into the cytosol, thus inducing cell death in AML cells [154]. Similarly, cationic-amphiphilic antihistamines also target leukemia cells in patients [158]. In another strategy, overexpression of p53-inducible gene 7 (pig7) in AML cell lines made them more susceptible to VP16 (etoposide) and daunorubicin combined treatments, thus inducing cell death [159].

Another approach for treating AML is by targeting the V-ATPase proton pump on the lysosome membrane, which maintains lysosomal acidity. While V-ATPase overexpression leads to chemoresistance, its knockdown made doxorubicin-resistant breast cancer (MCF-7) cells more sensitive to doxorubicin and vincristine combined [160]. Archazolid A, a V-ATPase inhibitor, has been reported to have an anti-leukemic effect by suppressing lysosomal acidification [161,162].

On the other hand, a microtubule inhibitor, deoxysappanone B 7, 4′-dimethyl ether (Deox B 7, 4), showed anti-leukemic activity by augmenting lysosomal V-ATPase activity, resulting in hyper-acidification of lysosomes and induced apoptosis of AML cells [161]. Cationic amphiphilic drugs (CAD) are another category of small molecules that accumulate in lysosomes and induce lysosomal cell death in multiple AML cell lines by altering the lipid profile in the lysosomal lumen. A few examples of these CAD are the antihistamines desloratadine, ebastine, loratadine, astemizole, and terfenadine; the antimalarials chloroquine and mefloquine; and the antidepressants desipramine, penfluridol, and siramesine [163]. Quercetin flavonoid, a polyphenol compound, induces lysosomal cell death in leukemia cells [164].

It has been shown that Dp44mT, a metal chelator that accumulates in the lysosomes, induces LCD [165]. In another study, Dp44mt induced the release of CTSD from the lysosomes into the cytosol and initiated mitochondrial cytochrome-c-dependent apoptosis [166,167]. Several other amphiphilic drugs, such as tricyclic antidepressants and antihistamines, impaired lysosomal membrane integrity and induced apoptosis via acid sphingomyelinase inhibition in AML cells [155]. In summary, lysosomal acidification plays a role in various disorders, and appropriate targeted approaches may be selected based on lysosomal acidification status.

Targeting lysosome signaling

Numerous mTORC1 inhibitors have been developed (Figure 5), including rapamycin and its analogs, targeting mTORC1, mTORC2, and PI3K. Phase I/II trials using mTORC1 inhibitors combined with chemotherapy have shown encouraging results in AML [168]. AZD2014 dual inhibitor of mTORC1/2 also reduced lysosomal pH in AML cells, increasing the cytotoxicity of the antibody-drug conjugate gemtuzumab ozogamicin (GO) [169]. It is good to use an antibody conjugate like GO in AML because most AMLs over-express CD33. GO needs an acidic environment inside lysosomes to hydrolyze the linker molecule and induce apoptosis in cancer cells. mTORC1-associated protein Raptor is a potential target in AML because it inhibits leukemia development with no effect on LSC self-renewal [170]. In mixed-lineage leukemia (MLL), inhibition of mTORC1 makes cancer cells more sensitive to lysine-specific demethylase 1 inhibitors and induces differentiation in MLL leukemia [171]. LAMP5 acts as an autophagy suppressor, so targeting it makes it easier for AML cells to get rid of the MLL fusion protein, which aids the survival of AML [172]. Four-amino-2-trifluoromethyl-phenyl retinate (ATPR), a new ATRA derivative, showed anticancer activity towards AML by initiating ferroptosis in AML cancer cells via a mechanism involving macroautophagy [173]. ATRA treatment inhibited fatty acid synthase (FASN) expression and facilitated differentiation of APL cells to granulocytes by translocating TFEB to the nucleus, which increases lysosomal biogenesis and autophagy [174].

Another study showed that g-interferon-inducible lysosomal thiol reductase (GILT) inhibition enhances AML chemosensitivity by elevating reactive oxygen species (ROS) levels and inducing oxidative mitochondrial-damage-mediated apoptosis. Similarly, inhibition of the PI3K/Akt/NRF2 antioxidant pathway enhanced the intracellular oxidative state and increased the chemosensitivity of AML [175].

Lysosomal cathepsins

Lysosomal CTS are the enzymes which help cancer progression, metastasis, angiogenesis, and chemoresistance [12,176]. CTS expression and activities are often upregulated in leukemia and solid tumors, such as melanoma, breast cancer, and gastrointestinal cancer [177,178,179]. For example, the nonreceptor tyrosine kinases Abl and Arg (Abl/Arg) have been shown to induce CTS B and CTS L secretion, which promote melanoma invasion and metastasis by degrading extracellular matrix proteins [180]. CTS have been suggested as targets for the treatment of multiple cancer types, including AML [177,181,182,183].

Stefin A and cystatin C are endogenous reversible CTS inhibitors with therapeutic potential in cancer [177,182]. Multiple strategies are available to target CTS, such as chemical inhibitors including CA074, odanacatib (MK-0822), KGP94, CLIK-148, and CLIK-195, which are highly specific and efficient; and CTS antibodies to target CTS secretion, which hold considerable potential for cancer treatment [12,129,177,180,184].

Although multiple options are available to target CTS, only a few of them could reach clinical trials. CTS are hard to target for clinical use because of their complexity [176,177,180,185]. In addition, enzyme replacement therapy is not yet well-established, and it is hard to deliver CTS accurately and efficiently to specific organs.

Other therapeutic strategies for AML

A more recent understanding of autophagy and/or lysosome dysfunction has given rise to numerous novel therapeutic techniques tested in clinical trials. Due to a strong correlation between lysosomal dysfunction and reduced immunological signals in the cancer immune response, increasing attention has been dedicated to enhancing the cancer immune response by lysosomal dysfunction [72,186]. A novel approach to target AML cells involved the development of a biohybrid with tumor-targeting peptide somatostatin and photosensitizer ruthenium (RU-SST). Somatostatin receptor type 2 (SSRT2) is highly expressed on AML cell lines and leukemic cells of AML patients compared to HSCs of healthy donors, thus increasing the selectivity of RU-SST to AML cells. As a result, the somatostatin biohybrid targets AML cells more effectively. Remarkably, RU-SST is found in lysosomes, suggesting that they are involved in the degradation mechanism [187]. Another novel strategy, namely non-thermal plasma (NTP), involves an ionized gas made up of excited atoms and molecules. NTP is known to induce apoptosis in AML cell lines, HL60, and KG-1 by suppressing lysosome activity [188]. The polyketal-based delivery of cytarabine is also an effective alternative therapeutic strategy for AML [189].

CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) protein colocalized with programmed death-ligand 1 (PD-L1) and prevented its lysosomal degradation, which evaded T-cell-mediated immunosurveillance, leading to immunological escape. In another study, the interaction of Huntingtin-interacting protein-1-related protein (HIP1R) with the conserved domain (771–867) of PD-L1 enhanced the lysosomal degradation of PD-L1 by directing its transportation to the lysosomes [190]. PD-LYSO is a peptide with the PD-L1-binding sequence, and the lysosome sorting sequence of HIP1R in it showed increased lysosomal degradation [190]. Furthermore, an aloperine derivative SA-49 increased lysosome biogenesis and MITF-dependent lysosomal degradation of PD-L1 in non-small-cell lung cancer cells [191]. SA-49 enhances the immune response of cocultured T and NK cells against cancer cells and inhibits the development of Lewis tumor xenografts [191]. Autophagy degrades MHC-I, and scientists have explored integrating immune checkpoint blocking (ICB) treatment with autophagy suppression [192]. In mice with orthotopic tumors, the combination of chloroquine (CQ) with anti-PD1 and anti-CTLA-4 antibodies resulted in an increased anti-tumor immune response [192].

TFEB, which controls the lysosomal–autophagic pathway, has been shown to be efficient in delaying the advancement of lysosome-related disorders such as cancer [193]. Furthermore, a recent study highlighted several chemicals discovered in recent years that influence the expression or nuclear translocation of TFEB, including 3,4-dimethoxychalcone (3,4-DC), 2-hydoxypropyl-cyclodextrin (HPCD), and digoxin [194,195].

CMA has been implicated in the growth of cancer [196], and it has been demonstrated that silencing LAMP2A reduces cancer cell proliferation and decreases transcription of heat shock cognate protein 70 (HSC70); moreover, knocking out of LAMP2A exacerbates the accumulation of pathological proteins associated with neurodegenerative diseases such as -synuclein, mutant huntingtin (mHTT), and Tau [196]. Additionally, a peptide, P140, was shown to suppress CMA in lupus-prone animals by downregulating the expression of both LAMP2A and HSC70 and decreasing HSC70 protein folding [75]. CID1067700, a Rab7 GTPase receptor antagonist, has been shown to decrease reactive astrogliosis and reduce brain shrinkage in astrocytic injury models by reducing excessive CTSB translocation from endosomes to lysosomes [197].

Adult T-cell leukemia/lymphoma (ATL) is a highly chemo-resistant malignancy of peripheral T lymphocytes caused by human T-cell leukemia virus type 1 infection [198]. A novel HSP90 inhibitor, TAS-116 (pimitespib), inhibits tumor cell growth in an animal model of ATL. NVP-AUY922 (AUY922), an HSP90 inhibitor, inhibited the in vitro growth of both primary and ATL cell lines [199].

Interestingly, lysosomes are being used as biologically derived nanoparticles. It is possible to encapsulate cancer drugs in nanoparticles such as liposomes. Lysosomes are ideal carriers due to their biological environment stability and minimal immunogenicity. For example, yeast-derived lysosomes carrying daunorubicin were effective against the HL60 AML cell line [200]. Targeting lysosomes in HSCs as well as in AML is still in its early stages. A further detailed investigation in lysosomal research could lead to significant advancement in the field.

15. Conclusions

Lysosomes are an important component of normal cellular homeostasis and affect a vast array of diseases and cellular states such as infections, metabolic disorders, and multiple cancer types, including AML. Lysosome activity, number, and size were found to be important factors for HSCs quiescent state maintenance. A recent study showed that the activated but not quiescent HSCs rely on glycolysis for energy [102]. Further, higher lysosomal activity was correlated with the active state of the HSC. Thus, aberrant activation of lysosomes could increase glycolysis and fulfill high energy demands of cancer cells, including AML cells. Lysosomes hold a potential for cancer therapy, and with newer classes of anti-cancer agents that utilize lysosomes, cancer therapeutic approaches could be tailored to become more tumor-specific with less off-target toxicities [201]. Cytarabine is one of the few recognized therapies for AML in younger people when combined with anthracyclines or stem cell transplantation, but it is ineffective in older patients [202,203]. Therefore, targeted treatments, which are less stringent in general therapy, may be very successful as monotherapy. Next, we focused on lysosomal functions, which may be the targets of future treatment for AML, particularly since their effects may be significantly enhanced when coupled with other modalities of targeted therapy. While the importance of autophagy in AML is obvious, the results are contradictory, perhaps because the process has different effects at different phases of leukemic transformation.

Further studies are required in order to fully understand the lysosome signaling capabilities, which have become associated with its degradative activities in recent years. This review also highlights and provides opportunities for developing novel means of intervention in myeloid malignancies. Lysosomes may also be used as a novel approach for identifying LSCs. However, more extensive research is required to determine whether or not lysosomes regulate stem cell quiescence in leukemia, and whether they are altered in aging HSCs, as they are in aged neuro-stem cells. Therefore, lysosomes may provide better means to maintain and expand HSCs in culture for bone marrow transplantation, which is currently a significant challenge.