Autophagy as a pathogenic mechanism and ... - The FASEB Journal

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Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders Marina Pierdominici,* Cristiana Barbati,*,‡ Marta Vomero,* Silvia L. Locatelli,§,储 Carmelo Carlo-Stella,§,储 Elena Ortona,*,¶,1 and Walter Malorni†,¶,1,2 *Department of Cell Biology and Neurosciences and †Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanita`, Rome, Italy; ‡Istituto San Raffaele, Sulmona, Italy; § Department of Oncology and Hematology, Humanitas Cancer Center–Humanitas Clinical and Research Center, Rozzano, Italy; 储Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy; and ¶Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), San Raffaele Pisana, Rome, Italy Autophagy represents a key mechanism of cytoprotection that can be activated by a variety of extracellular and intracellular stresses and allows the cell to sequester cytoplasmic components and damaged organelles, delivering them to lysosomes for degradation and recycling. However, the autophagy process has also been associated with the death of the cell. It has been demonstrated to be constitutive in some instances and inducible in others, and the idea that it could represent a pathogenetic determinant as well as a possible prognostic tool and a therapeutic target in a plethora of human diseases has recently been considered. Among these, cancer represents a major one. In this review, we recapitulate the critical implications of autophagy in the pathogenesis, progression, and treatment of lymphoproliferative disorders. Leukemias and lymphomas, in fact, represent paradigmatic human diseases in which advances have recently been made in this respect.—Pierdominici, M., Barbati, C., Vomero, M., Locatelli, S. L., Carlo-Stella, C., Ortona, E., Malorni, W. Autophagy as a pathogenic mechanism and drug target in lymphoproliferative disorders. FASEB J. 28, 524 –535 (2014). www.fasebj.org

ABSTRACT

Abbreviations: AMPK, AMP-activated protein kinase; Atg, autophagy related; Bcl-2, B-cell lymphoma 2; BCR, B-cell receptor; CLL, chronic lymphocytic leukemia; CQ, chloroquine; DLBCL, diffuse, large B-cell lymphoma; Env, envelope; ERK, extracellular signal-regulated kinase; FADD, Fas-associated protein with death domain; FL, follicular lymphoma; HDAC, histone deacetylase; HL, Hodgkin’s lymphoma; HRS, Hodgkin and Reed-Sternberg; IgVH, immunoglobulin heavychain variable-region; LC3, microtubule-associated protein 1 light chain 3; MCL, mantle cell lymphoma; miRNA microRNA; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NF-␬B, nuclear factor ␬B; NHL, non-Hodgkin’s lymphoma; PI3K, phosphatidylinositol 3-kinase; PKC␨, protein kinase C␨; PTEN, phosphatase and tensin homolog; RTX, rituximab; SLE, systemic lupus erythematosus; Syk, spleen tyrosine kinase; TCR, T-cell receptor; TRAIL, TNFrelated apoptosis-inducing ligand; TSC, tuberous sclerosis complex; ZAP-70, ␨-chain-associated protein kinase 70 kDa 524

Key Words: cancer 䡠 lymphocytes 䡠 Hodgkin’s lymphoma 䡠 nonHodgkin’s lymphoma 䡠 cell fate Lymphoid malignancies encompass a heterogeneous group of B-, T-, and NK-cell neoplasms with a remarkable ability to adapt to cytotoxic or proapoptotic drugs (1–3). Hodgkin’s lymphoma (HL) accounts for ⬃10% of all lymphomas, and the remaining 90% are referred to as non-Hodgkin’s lymphoma (NHL). HL involves peripheral lymph nodes and can also affect a variety of extranodal sites, including liver, lung, and bone marrow. The HL-generating tumor cell, referred to as the Hodgkin and Reed-Sternberg (HRS) cell, is very rare and usually accounts for only 1–5% of the cells in the tumor mass. The remainder are a mix of reactive inflammatory cells attracted by the HRS cells. Although HRS cells are derived from mature B cells, they have largely lost their B-cell phenotype and show a very unusual coexpression of markers of various hematopoietic cell types. NHL encompasses a heterogeneous group of cancers, 85–90% of which arise from B lymphocytes and the remainder from T or NK cells. These malignancies usually develop in the lymph nodes, but can occur in almost any tissue and range from the more indolent follicular lymphoma (FL) to the more aggressive diffuse, large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma. The standard of care for each type of lymphoma can vary; most regimens encompass combinations of cytotoxic drugs together with monoclonal antibodies. Because of the inherent heterogeneity of the lymphomas, there are very different responses to treatments, and, although the survival rate of the patients has improved over the past decades, many patients with aggressive lymphomas do not show a durable response to treatment. Moreover, many of the first-line chemotherapy regimens are too toxic to be 1

These authors contributed equally to this work. Correspondence: Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanita`, Rome, Italy. E-mail: [email protected] doi: 10.1096/fj.13-235655 2

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tolerated by the elderly. Therefore, novel approaches to the treatment of lymphomas are needed. Of particular interest are molecularly targeted therapies that interfere with the critical signal transduction pathways that determine cell survival and proliferation. These signal transduction pathways can be activated through interactions of tumor cells with other cell types detectable in the microenvironment, but also through genetic defects [e.g., nuclear factor-␬B (NF-␬B), Janus kinase/signal transducer and activator of transcription (JAK/STAT); notch, mitogen-activated protein kinase (MAPK); and phosphatidylinositol 3-kinase (PI3K)/ Akt/mammalian target of rapamycin (mTOR) pathways; refs. 4, 5]. In this context, autophagic machinerytargeted agents are emerging as promising tools for treatment of lymphoma (6 –9). Macroautophagy (hereinafter referred to as autophagy) is a highly conserved, lysosome-mediated, catabolic process that allows cells to degrade, in a regulated manner, unwanted cytoplasmic constituents and recycle nutrients (10). It is morphologically characterized by the formation of doublemembrane vesicles (autophagosomes) that sequester cytoplasmic components and deliver them to lysosomes for degradation and recycling. In addition to its function as a survival mechanism during nutrient starvation, autophagy has an important role in many biological processes, including cell development, metabolism, immunity, and aging (10). Autophagy also has an indirect or direct role in cell death, and interactions between regulatory elements of both autophagy and apoptosis suggest a complex crosstalk between these two processes (11). However, the mechanism regulating autophagic cell death and its crosstalk with apoptosis remains to be better characterized (11). Of note, dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including hematological malignancies (6, 12). Herein, we briefly review the molecular mechanisms of autophagy regulation and the role of autophagy in lymphocyte homeostasis. Next, we cover the latest developments concerning autophagy’s influence on the initiation and progression of lymphoid malignancies and conclude by presenting new therapeutic approaches that target autophagy in the treatment of these disorders focusing on HL and NHL.

AUTOPHAGY REGULATION Autophagy is a very complex process controlled by the autophagy-related (Atg) genes described in detail in several elegant and exhaustive reviews (13–15). In this article, only signals of particular relevance to lymphomagenesis will be described briefly (Fig. 1). The initial signal to form autophagosomes is instructed by the Beclin-1-interacting complex, consisting of Beclin-1/ Atg6, class III PI3K (hVps34), the regulatory protein kinase p150 (hVps15), and Atg14L. Activation of this complex generates phosphatidylinositol 3-phosphate, which promotes autophagosomal membrane nucleAUTOPHAGY IN LYMPHOPROLIFERATIVE DISORDERS

Figure 1. Autophagy signaling pathways involved in lymphomagenesis and molecularly targeted agents under clinical investigation. The autophagy-related lymphoma network mainly involves the PI3K/Akt/mTOR pathway, the Beclin-1 complex, and p53 signaling that may be potential novel drug targets for lymphoma treatment. The circled names of molecules on a white background represent the factors that stimulate autophagy, whereas those on a gray background correspond to inhibitory factors. The names of anticancer agents currently under investigation in clinical trials are shown in the rectangles. HCQ, hydroxychloroquine.

ation. This process is negatively regulated by the binding of B-cell lymphoma 2 (Bcl-2) family members, such as Bcl-2 and Bcl-XL, to Beclin-1, preventing Beclin-1 from binding to the PI3K complex and thereby inhibiting autophagy (16). The constitutive Bcl-2/Beclin-1 interaction is disrupted by signals that promote autophagy. For example, in response to starvation or ceramide treatment, c-Jun NH2-terminal kinase 1 phosphorylates Bcl-2 to trigger its release from Beclin-1, inducing autophagy. Vesicle elongation and completion are mediated by two ubiquitin-like systems: the microtubule-associated protein 1 light chain 3 (LC3) and Atg12–Atg5. Autophagosome formation is negatively regulated by the mTOR pathway, a nutrientsensing kinase pathway that acts as a key regulator of cell growth, survival, metabolism, and proliferation (17–19). mTOR is the catalytic subunit of two distinct complexes called mTOR complex 1 (mTORC1) and mTORC2. mTORC1 contains mTOR and regulatoryassociated protein of mTOR (Raptor), and the mTORC2 complex contains mTOR and rapamycininsensitive companion of mTOR (Rictor). The rapamycin-sensitive mTORC1 complex promotes mRNA translation and inhibits autophagy by integrating different signals that are generated by growth factors, energy depletion, and various stressors, including hypoxia and 525

DNA damage. The regulatory role of mTORC2, which is less sensitive to rapamycin, is only partially known, but the available evidence suggests that it can facilitate actin cytoskeleton rea mTORC2 are inactivated, the processes involved in cell growth and proliferation are inhibited, and autophagy can take place. A major signaling cascade controlling mTORC1 is the class I PI3K/Akt pathway. In response to insulin and other growth factor signaling, the activation of class I PI3K leads to phosphorylation and activation of Akt. In turn, Akt phosphorylates and inhibits the mTOR repressor tuberous sclerosis complex 2 (TSC2; tuberin), which forms the TSC with TSC1 (hamartin), thus leading to activation of mTORC1. mTORC1 negatively regulates the induction of autophagy by phosphorylating and inactivating unc-51-like kinase 1 (ULK1), a proximal component of the autophagy signal transduction cascade. Akt may also negatively regulate autophagy by phosphorylating Beclin-1/Atg6. In addition to the PI3K/Akt pathway, growth factor signaling may activate extracellular signal-regulated kinase (ERK), which phosphorylates TSC2, at different sites from Akt, resulting in the inhibition of its activity. Conversely, a decrease in the normally high intracellular ATP:AMP ratio leads to AMP-activated protein kinase (AMPK) activation, which negatively regulates mTOR, acting as a positive regulator of autophagy. Metabolic stress also inhibits mTORC1 activity through p53-dependent upregulation of negative regulators, such as AMPK, TSC1, and TSC2. Note that p53 plays a complex role in the control of autophagy (20). In fact, on the one hand, nuclear p53 induces autophagy by transactivating mTOR inhibitors and several metabolism genes; on the other hand, cytoplasmic p53 inhibits autophagy by promoting the mTOR pathway. As detailed below, many of the signaling pathways described so far are shared between activated lymphocytes and lymphoma cells (e.g., the PI3K/Akt/mTOR pathway; ref. 21). Studies on the transition from naive to activated cells can help in learning about the pathways involved in this shift and to define new possible autophagy-related therapeutic targets in lymphoid cancers.

AUTOPHAGY AND LYMPHOCYTE DEVELOPMENT AND ACTIVATION Freshly isolated cells, particularly lymphocytes, although of great interest in translational medicine, represent a very complex cell type for the study of autophagy. In fact, they display a series of pitfalls due to, among others factors, the simultaneous presence of different subpopulations with various features and functions; the activation processes, which modify their function; the difficulties in culturing these cells; and the peculiar, very high nuclear/cytoplasmic ratio (i.e., the very small cytoplasmic milieu). Hence, the study of autophagy in primary lymphocytes has developed only during the past few years. These investigations, which 526

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included the analysis of specific knockout or transgenic models, revealed that autophagy plays an essential role not only in maintaining intracellular homeostasis, but also in sustaining, during lymphocyte development, a healthy self-renewing population of cells, not susceptible to leukemia transformation, capable of populating the T- and B-lymphocyte compartments (22). Autophagy and T lymphocytes Inactivation (or loss) of autophagy-related genes, such as Atg5, Atg7, and Beclin-1, reduces thymic cellularity, suggesting the crucial role of autophagy in normal lymphopoiesis (23–26). In the same vein, as concerns the peripheral T-cell compartment, Atg5-, Atg7-, and Atg3-defective T lymphocytes display impaired proliferation due to T-cell receptor (TCR) stimulation, increased mitochondrial mass, and decreased cell survival. Conversely, peripheral cells appear to be unaffected by Beclin-1 deficiency (23–25, 27). Autophagosomes can be detected within freshly primary murine T lymphocytes, and their number and size increase on withdrawal of serum, amino acids, or TCR stimulation (23, 28, 29). Autophagy is upregulated in Th2 CD4⫹ T cells compared with Th1 CD4⫹ T cells (28). In addition, cell cultured in Th1 polarizing conditions rely more heavily on autophagy for survival than does the Th17 subset (30). These findings in murine T cells hold true for human T cells. Basal autophagy levels have been found to be higher in memory T cells than in naive T cells (31), and stimulation of CD8⫹ peripheral blood T lymphocytes results in a significant induction of autophagy (32). The results presented so far suggest a critical role for autophagy in T-lymphocyte survival, activation, and proliferation, and this effect has been attributed to autophagy’s maintaining mitochondrial turnover and degrading essential components of the apoptotic cell death machinery (33). In this regard, Kovacs et al. (30) found that Beclin-1-deficient CD4⫹ T cells, which lack autophagy, are prone to apoptosis on TCR stimulation, because of greatly elevated levels of cell death–related proteins, such as procaspase-3, procaspase-8, and Bim. In addition, autophagy may support the early stages of T-cell activation, when extracellular nutrition is limited, but metabolic reprogramming (i.e., the switch from catabolic to anabolic metabolism) for proliferation has already begun. However, besides its prosurvival function, autophagy may paradoxically be involved in T-cell death (34, 35). For instance, T cells lacking Fas-associated protein with death domain (FADD) or caspase-8, both essential for death receptor–induced apoptosis, succumb to the hyperactivation of autophagy and die through a nonapoptotic form of cell death (i.e., necroptosis) rather than proliferating after mitogen stimulation (34). Furthermore, Espert et al. (35) found that autophagy is highly induced by the binding of the HIV envelope (Env) glycoproteins to the chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) on the surface of

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CD4⫹ T cells. Notably, Env-mediated autophagy is necessary to trigger CD4⫹ T-cell apoptosis, shown by the fact that blockade of autophagy totally inhibits the apoptotic process. Furthermore, CD4⫹ T cells still undergo Env-mediated cell death with autophagic features when apoptosis is inhibited. Autophagy and B lymphocytes Autophagy is also needed for B-lymphocyte development. Loss of Atg5 and Beclin-1 results in a significant defect in B-lymphoid progenitors (23, 26, 36). Of interest, Sin1⫺/⫺ immature B cells exhibit some key defects, including increased V(D)J recombinase activity, suggesting a role for Sin1/mTORC2 signaling in preventing aberrant V(D)J recombination, which may result in genomic instability. These findings raise the possibility that drugs that inhibit mTOR favor genomic instability by promoting aberrant V(D)J recombinase activity. Furthermore, B-cell loss of the negative regulator TSC1 leads to a significant reduction in marginal zone B cells, which is partially corrected by rapamycin administration (37). A mouse phenotype with a hypomorphic allele of mTOR that reduces mTOR protein expression and diminishes the activity of both mTORC1 and mTORC2 has recently been described (38). These mice have a partial block in early B-cell development, a reduced number of peripheral B cells, and impaired proliferative responses to anti-IgM or anti-CD40. Watanabe et al. (39) showed that B-cell receptor (BCR) ligation (cross-linking of BCR by either the ligand, i.e., the antigen, or by antibodies that react to BCR) induces the accumulation of autophagosomes in both primary B cells and B-cell lines. Interestingly, extensive autophagosome formation is induced when BCR-ligated B cells undergo apoptosis, and it is blocked when costimulatory signaling through CD40 rescues the B cells from apoptosis, suggesting that autophagy is induced by apoptotic BCR signaling. In an attempt to better define the functional relevance of autophagy in B-lymphocyte lineage (e.g., in plasma cells), Pengo et al. (40) recently demonstrated that autophagy is strongly induced after B-cell activation and is necessary to maintain the long-lived memory plasma cell pool in the bone marrow. Lack of autophagy was instead toxic to plasma cells, causing a substantial increase in cell death. In sum, all these findings indicate that the role of autophagy is dependent on the cell type and the stimulus and that blocking autophagy can skew the balance of immune subsets. The notion that regulation of cell death protein turnover by autophagy is a critical self-limiting mechanism in activated lymphocytes is relevant, not only to the pathogenesis of malignant lymphoid disorders, but also to that of nonmalignant lymphoproliferative disorders, particularly autoimmune disease in which the persistence of autoreactive T- and B-cell clones is a typical feature. In this regard, our recent data suggest that chronic exposure to specific autoantibodies, as occurs in systemic lupus eryAUTOPHAGY IN LYMPHOPROLIFERATIVE DISORDERS

thematosus (SLE), could lead to the selection of autophagy-resistant autoreactive T lymphocytes (31). In this context, the failure of autophagy induction could also result in an overload of damaged mitochondria, release of apoptogenic factors from these organelles, and excessive reactive oxygen species production, events that are commonly observed in SLE. Supporting this observation, Fernandez et al. (41) found that rapamycin reduces disease activity and normalizes Tcell activation in patients with SLE, probably restoring the autophagic response. Autophagy and lymphomagenesis The influence of autophagy on the initiation and progression of cancer as well as on the effectiveness of therapeutic interventions appears to be multifaceted (42). Particularly in the early stages of cancer development, autophagy provides an anticarcinogenic function by safeguarding against metabolic stress through the homeostatic turnover of mitochondria and the clearance of protein aggregates. Genetic deletion of autophagy proteins causes mitochondrial dysfunction, increased oxidative stress, and susceptibility to proinflammatory stimuli, conditions that permit DNA damage. In turn, this harm can lead to genetic instability and facilitates or even triggers tumorigenesis. Autophagy can also play a role in the maintenance or entry of cells into the G0 phase of the cell cycle, and, consequently, it can prevent spontaneous hyperproliferation of cells. In contrast, in established tumors, autophagy may enable tumor cells to tolerate stress, including a hypoxic microenvironment, nutrient deprivation, and probably some forms of therapy. Even with prolonged stress, autophagy supports cell survival, generating “dormant” tumor cells that have the capability of resuming growth when conditions are more favorable (43). The process of stress resistance, dormancy, and regeneration afforded by autophagy may be a major obstacle to achieving successful cancer treatment. Although a prosurvival role for tumor autophagy during chemotherapy is generally accepted, whether autophagy facilitates chemotherapeutic- or radiationinduced cytotoxicity in apoptosis-resistant tumor cells through autophagy-associated cell-death pathways has yet to be clarified. Herein, we highlight key discoveries and controversies on how the functional status of autophagy influences lymphomagenesis and treatment response. Defective autophagy as a mechanism of lymphomagenesis Studies in mouse models have assigned a tumor-suppressor function to core autophagy proteins and autophagy-associated proteins. Mice heterozygous for Beclin-1 (Beclin-1⫹/⫺) show reduced autophagy and increased cell proliferation, which result in a high incidence of spontaneous malignancies, such as lymphomas (44). Knockout of Bif-1, which forms a com527

plex with Beclin-1 and facilitates autophagosome formation under starvation conditions, promotes spontaneous tumorigenesis, and Bif-1⫺/⫺ mice have a significantly higher lymphoma incidence than do wild-type mice (45). Other data refer to the Atg5 protein, a ubiquitin ligase that has a role in autophagosome elongation. Atg5 protein is involved in IFN-␥-induced autophagic cell death by interacting with FADD (46), and its low expression has been hypothesized to favor survival of tumor cells. In fact, haploinsufficiency of Atg5 has been suggested to promote lymphoid malignancies (47). In addition to the direct evidence that defective expression of autophagy proteins promotes lymphomagenesis, there is also a strong overlap between activation of signaling pathways leading to tumorigenesis (e.g., upstream regulators in mTOR signaling) and suppression of autophagy. As stated earlier, the PI3K/ Akt axis plays a decisive role in the negative regulation of autophagy by stimulating mTOR, and the activation of this pathway is observed in several lymphoid malignancies, including HL, mantle cell lymphoma (MCL), DLBCL, chronic lymphocytic leukemia (CLL), FL, and anaplastic large-cell lymphoma (7, 9, 48, 49). Loss of expression of the protein product of the tumor suppressor gene phosphatase and tensin homolog (PTEN), which negatively regulates PI3K, or its constitutive inactivating phosphorylation, is a common feature in HL (50) and in MCL (51). Other mechanisms may account for mTOR activation, such as the tonic (i.e., low level constitutive) antigen-independent BCR activation that has been observed in several B-cell lymphomas (9). Particularly, spleen tyrosine kinase (Syk), which is rapidly phosphorylated after BCR activation, appears to contribute to mTOR-positive regulation through an unknown PI3K-independent pathway, and it has been found to be overrepresented in several lymphomas (e.g., FL, DLBCL, MCL, and Burkitt’s lymphoma). Syk inhibition by a chemical inhibitor or small interfering RNA, results in a potent inhibition of mTOR activity in these B-cell lymphomas (52). A further regulatory mTOR pathway has been demonstrated in FL cells, consisting of a protein kinase C␨ (PKC␨)/ERK/mTOR module (53). PKC␨, an atypical form of PKC, displays increased expression and activity in FL cells and appears to be a significant contributor of abnormal mTOR regulation through an ERK-dependent mechanism. Of interest, rituximab (RTX), an anti-CD20 mouse/human chimeric IgG1␬ antibody used in the treatment of various lymphoid malignancies, has been found to selectively inhibit PKC␨/ERK/mTOR in FL cells, but not in DLBCL and MCL cells. Moreover, combined in vitro treatment of FL cells with RTX and rapamycin result in an additive antileukemic effect, suggesting that this association represents a new therapeutic strategy. Alternatively, Shi et al. (54) have recently found that AMPK activity is completely lost in both T- and B-cell lymphoma cells and is related to the up-regulation of the mTOR signaling pathway. Of note, the AMPK activator metformin, an oral hypoglycemic agent, was able to modulate the AMPK/mTOR axis, 528

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inducing AMPK activation and consequent inhibition of the mTOR signaling, resulting in lymphoma cellgrowth inhibition and drug sensitization. Overall, mTOR’s apparent regulation by several distinct intracellular signaling pathways designates this kinase as an important molecular target for lymphoma treatment (7, 9, 48, 49). Recent data on the implication of microRNA (miRNA)— endogenously expressed, noncoding RNA—in lymphomagenesis should be reported. In fact, growing evidence suggests that altered regulation of target oncogenes or tumor suppressors by miRNA contributes to malignant lymphoma (55). For example, miRNA15a/16-1, which negatively regulates Bcl-2, is deleted or down-regulated in most cases of CLL (56), whereas miRNA-17–92, which dampens the expression of PTEN, is highly expressed in aggressive B-cell lymphoma (57). Incidentally, in human lymphoblastoid cell lines, miRNA-15a/16-1 is transactivated by the tumor suppressor p53, whereas the effect of this protein on the miRNA-17–92 cluster is less clearly defined (58). Enhanced autophagy as a mechanism of lymphoma cell survival All these studies support a role for autophagy in tumor suppression. However, other studies support the hypothesis that autophagy correlates with tumor progression and drug resistance in lymphoid malignancies. For example, Valentin-Vega et al. (59) found that, in ataxia– telangiectasia mutant null mice, Beclin-1 heterozygosity delayed, rather than promoted, T-cell lymphomagenesis, thus suggesting that Beclin-1 cannot be considered as merely a tumor-suppressor gene, because its effect on cell fate is context dependent. However, in view of this finding, the most important question is whether autophagy-hindering drugs represent a new promising therapeutic approach. For instance, a key compound capable of hindering autophagy and fighting tumor progression is the antimalarial drug chloroquine (CQ), which raises lysosomal pH and blocks the degradation of sequestered material in autophagosomes. In a mouse model of lymphoma, inhibition of autophagy through the administration of CQ impaired tumor formation and enhanced survival (60). Accordingly, another study performed several years ago in humans demonstrated that malarial prophylaxis with CQ decreases the incidence of Burkitt’s lymphoma (61). If these works provide preclinical evidence that CQ could be effective in the prevention of lymphoma, other studies also suggest that inhibition of autophagy could be relevant in cancer treatment. In fact, as tumor cells exposed to chemotherapeutic agents undergo autophagy to support survival by increasing bioenergetics (42), it is not surprising that the use of CQ in combination with standard anticancer treatments correlates with increased antitumor activity. For instance, Amaravadi et al. (62) demonstrated that in a Myc-induced model of lymphoma, inhibition of autophagy by either CQ or short-hairpin RNA for Atg5, enhanced the ability of


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alkylating drug therapy to induce tumor cell death. In the same vein, Amrein et al. (63) found that dasatinib, a tyrosine kinase inhibitor currently investigated in the treatment of CLL, induces p53-mediated autophagy and, consequently, drug resistance in CLL lymphocytes, and that CQ sensitizes tumor cells to dasatinib. More recently, Sommermann and colleagues (64) demonstrated that the autophagy inhibitors 3-methyladenine and CQ are effective when associated with NF-␬B inhibitors in B-cell lymphoma. In fact, the pathway associated with this transcription factor promotes cell survival by increasing glucose import, and its constitutive activation is a common feature of transformed B lymphocytes. Thus, combined inhibition of autophagy and the NF-␬B pathway can drive the cells into metabolic crisis, accelerating cell death. Moreover, targeting TNF-related, apoptosis-inducing ligand (TRAIL) receptors with either recombinant TRAIL or agonistic specific antibodies has been considered a promising treatment for cancer, particularly in light of the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, many hematological malignancies, including CLL, are resistant to TRAIL treatment (65, 66). One reason for this resistance may be that TRAIL induces cytoprotective autophagy (67). Of note, knockdown of the autophagic genes Beclin-1 and Atg5 sensitizes leukemia cell lines to TRAIL-mediated apoptosis. These results provide the basis for a novel therapeutic approach to potentiate TRAIL efficacy by the inhibition of autophagy of tumor cells (67). Further actors contributing to the mechanisms of lymphoid cell survival by autophagy are miRNAs. A new miRNA–autophagy regulatory axis that modulates survival mechanisms in CLL cells has recently been suggested by Kovaleva et al. (68). They demonstrated that autophagy is a constitutive mechanism in CLL cells and that it is part of the stress response of these cells to the lack of nutrients. Notably, transfection of CLL cells with miRNA-130a, which is widely downregulated in these cells, reduces cell survival and inhibits autophagy, an effect partially mediated by down-regulation of the autophagic gene Atg2B, which is involved in the initial steps of autophagosome formation. Altogether, these data indicate that autophagy may

sustain tumor progression and drug-resistance and that it could represent an important target for treatment of lymphoproliferative disorders. However, the prosurvival function of autophagy, as observed for Beclin-1 (59), appears to be context dependent, so that whether to inhibit or activate autophagy, together with the timing for the treatment, remains a matter of debate.

AUTOPHAGY-RELATED PROGNOSTIC FACTORS The search for valuable prognostic markers in human diseases, including cancer, encompasses an extensive series of changes that occur at the periphery of or within the lesion. Soluble molecules in the peripheral blood from affected patients and alterations of circulating cells or specific changes in the affected organ are being considered. The milestone of these investigations is referred to as the identification of biomarkers that could be used as clinically valuable and solid indicators for making diagnoses, determining disease progression, or assessing response to therapy. Among the candidates, on the basis of its emerging role in cancer onset and progression, are autophagy and its subcellular determinants. In particular, several autophagy-related molecules have been taken into consideration in recent years, whose possible beneficial use in the clinical practice has been suggested in different forms of cancer (6, 69, 70). As concerns lymphoma, very few data have been provided so far; the available reports are summarized in Table 1. The level of Beclin-1 expression was demonstrated to predict clinical outcomes in patients with several solid tumors: increased expression was related to favorable prognoses in colon, ovarian, brain, and hepatocellular cancers and to unfavorable prognoses in nasopharyngeal, gastric, and colorectal cancers (77). As concerns lymphoproliferative disorders, the prognostic value of Beclin-1 in extranodal NK-/T-cell lymphoma has been analyzed by Huang et al. (71). In their study, low Beclin-1 expression was associated with worse overall survival and progressionfree survival. Similarly, DLBCL patients with high Beclin-1 expression had longer overall survival and were more responsive to standard treatments than were those with low Beclin-1 expression (72). A significant

TABLE 1. Autophagy-related molecules proposed as prognostic biomarkers in lymphoid malignancies Biomarker

Tumor type

Source

Beclin-1

ENKTL (71), DLBCL (72), NHL subtypes (73)

TS

LC3

Relapsed or refractory HL and NHL subtypes (74) CLL (75) CLL, DLBCL, HCL, MCL, and FL (76)

PB

miRNA15a/16-1 P53

PB PB, TS, leukemia/ lymphoma cell lines

Significance

Beclin-1 expression positively associates with overall survival, progression-free survival, and therapy response. High LC3 expression associates with good clinical outcome. miRNA signature associates with prognosis. P53 mutations associate with aggressive disease, short survival, and poor therapy response. Wild-type p53 associates with drug resistance.

ENKTL, extranodal NK-/T-cell lymphoma; HCL, hairy cell leukemia; LC3, microtubule-associated protein 1 light chain 3; PB, peripheral blood; TS, tissue specimens.

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correlation of Beclin-1 from bioptic specimens with the clinical outcome of NHL was also observed by Nicotra et al. (73). High levels of Beclin-1 aggregates were associated with a complete or partial remission, suggesting that NHL with upregulated autophagy can be more responsive to chemotherapy. Changes in the expression of LC3, one of the main markers of autophagy, have also been shown to correlate with malignant transformation. Similar to that observed for Beclin-1, expression of LC3 has been found to be significantly higher in the samples of benign and borderline ovarian tumors than in malignant epithelial ovarian cancers (78). In our recent study, baseline autophagy levels in peripheral blood lymphocytes from patients with relapsed or refractory HL and NHL, treated with the multikinase inhibitor sorafenib, were significantly higher in responsive patients than in nonresponsive patients (74). This suggests the involvement of autophagy in tumor progression, or even in tumor chemorefractoriness, and claims for the idea to boost the activity of kinase inhibitors by combining them with autophagy inhibitors. In addition, in the same study, a significant reduction in autophagy levels was found in blood lymphocytes from responsive patients, whereas no change was detected in the nonresponsive patients. The importance of these results resides in the possibility of optimizing selection of patients receiving targeted antilymphoma therapies by simply analyzing autophagy markers in the peripheral blood cells. Interesting data were also derived from the studies of miRNAs. A unique miRNA expression signature composed of 13 mature miRNAs, including miRNA-15a/ 16-1, has been found to differentiate cases of CLL with low levels of 70 kDa ␨-chain-associated protein kinase (ZAP-70) expression from those with high levels, and those with unmutated immunoglobulin heavy-chain variable-region (IgVH) from those with mutated IgVH (75). Note that cases in which the leukemic cells have few or no mutations in the IgVH gene or a high level of expression of ZAP-70 have an aggressive course, whereas cases involving mutated CLL clones or few ZAP-70 cells have an indolent course. The same miRNA signature has been associated with the presence or absence of disease progression. Regarding p53 mutations, their prognostic significance appears to be inconsistent in malignant lymphoma because of their puzzling role in cell death and cell survival (76). In fact, on the one hand, wild-type p53 can suppress lymphoma cell survival by inducing apoptosis, cell cycle arrest, and DNA repair, thus correlating with a better clinical outcome (e.g., in DLBCL, hairy cell leukemia, CLL, FL, and MCL); on the other hand, p53 can promote lymphoma cell survival by inducing autophagy. In this case, it is associated with a worse clinical outcome (e.g., in CLL). The mechanisms underlying the p53 functional switch from apoptosis to autophagy have not been elucidated, and their comprehension may help to modulate p53 function to promote, rather than protect against, lymphoma cell death. 530

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Overall, these findings suggest that the potency of the analyses of the autophagy-related molecules as prognostic markers could increase in the near future. Of note however, not only should bioptic materials be taken into account, but, much more intriguingly, even the peripheral blood of patients (e.g., those with lymphoma). These predictive tests could open novel and more tailored therapeutic strategies.

AUTOPHAGY AS A THERAPEUTIC TARGET Current therapeutic targeting of autophagy in cancer is limited by an incomplete understanding of how the autophagic process contributes to its pathogenesis; the lack of specificity of compounds that modulate autophagy; and the limited availability of candidate molecules with clinical efficacy. However, some anticancer agents acting as inducers of autophagy in lymphoid malignancies have already been tested in phase I/II clinical trials involving patients with relapsed or refractory lymphomas (Table 2). PI3K/Akt/mTOR inhibitors Promising preclinical and clinical data (ClinicalTrials. gov, U.S. National Institutes of Health, Bethesda, MD, USA: http://clinicaltrials.gov) support the rationale for the therapeutic use of PI3K/Akt/mTOR inhibitors in lymphoma (49). For instance, in preclinical studies using CLL cell lines and patient CLL cell samples, GS-1101 (CAL-101), a selective inhibitor of the PI3K isoform p110␦, blocked constitutive PI3K signaling, resulting in decreased Akt phosphorylation and cell viability (81– 83). These effects have been observed across a broad range of other B-cell malignancies, including MCL, DLBCL, and FL (83). Furthermore, cell cycle arrest and apoptosis by CAL-101 are induced in HL cell lines (84). Of interest, preliminary results from a phase I clinical trial on the safety of CAL-101, used as a single agent, have demonstrated overall response rates ranging from 26% to 86% in patients with relapsed or refractory NHL (ClinicalTrials.gov ID: NCT00710528). Other phase I/II clinical trials with CAL-101 in patients with relapsed or refractory HL or NHL are also ongoing (ClinicalTrials.gov ID: NCT01306643, NCT01393106, and NCT01282424). Combinations of CAL-101 with commonly used agents in B-cell NHL (i.e., RTX or RTX plus bendamustine) are also being tested in patients with indolent (slow-growing) relapsed or refractory NHL or older patients with untreated NHL (ClinicalTrials.gov ID: NCT01088048, NCT01732913, and NCT01732926). A further phase II clinical trial is ongoing on the efficacy, safety, tolerability and pharmacodynamics of CAL-101 and the Syk inhibitor GS-9973 in patients with relapsed or refractory NHL (ClinicalTrials.gov ID: NCT01796470). An important objective for such combination studies is to establish appropriate long-term dose levels and safety margins.


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TABLE 2. Examples of autophagy inducers of possible clinical relevance in lymphoid malignancies Class

PI3K inhibitors

Drugs

CAL-101

IPI-145 mTOR inhibitors (first generation)

Temsirolimus Everolimus

Ridaforolimus

mTOR inhibitors (second generation) HDAC inhibitors

CC-223

Multikinase inhibitors

Sorafenib

Vorinostat Panobinostat

Proposed use

Mechanism

Phase I/II trials as a single agent (ClinicalTrials.gov identifiers: NCT00710528, NCT01306643, NCT01393106, NCT01282424) or in combination therapy (ClinicalTrials.gov identifiers: NCT01088048, NCT01732913, NCT01732926, NCT01796470) Phase I clinical trial as a single agent (ClinicalTrials.gov identifier, NCT01476657) Authorized in Europe for MCL (79) Phase II clinical trial as a single agent (ClinicalTrials.gov identifier, NCT00436618) or phase I/II clinical trials in combination therapy (ClinicalTrials.gov identifiers: NCT00869999, NCT01334502, NCT01198665, NCT01665768, NCT01854606, NCT01088048, NCT00474929, NCT00869999, NCT01075321, NCT01567475, NCT00962507, NCT00352443, NCT01453504, NCT00935792, NCT01341834) Phase I/II clinical trials as a single agent (ClinicalTrials.gov identifiers: NCT00060632, NCT00060645, NCT00086125) or phase I clinical trial in combination therapy (ClinicalTrials.gov identifier, NCT01169532) Phase I/II clinical trial as a single agent (ClinicalTrials.gov identifier, NCT01177397)

Inhibition of PI3K-␦

Phase II clinical trial as a single agent (80) Phase I/II clinical trials in combination with everolimus (ClinicalTrials.gov identifiers: NCT00962507, NCT00918333, NCT00978432) Phase II clinical trial as a single agent (74)

Downregulation of Akt and mTOR signaling; upregulation of Beclin-1 and Atg7 expression

Inhibition of PI3K-␦ and PI3K-␥ Inhibition of mTORC1 Inhibition of mTORC1

Inhibition of mTORC1

Inhibition of mTORC1 and mTORC2

Inhibition of mTOR signaling

PI3K-␦, p110␦ isoform of PI3K; PI3K-␥, p110␥ isoform of PI3K.

In addition to CAL-101, promising early clinical data are available for IPI-145, a potent inhibitor of both the p110␦ and p110␥ isoforms of PI3K, which are involved in thymocyte survival (87) and in lymphocyte chemotaxis and homing (79). Preliminary results from an ongoing phase 1 clinical trial of IPI-145 (ClinicalTrials. gov ID: NCT01476657) show that this drug has a good tolerability and rapid clinical responses in patients with both B- and T-cell malignancies, including those with CLL, indolent NHL, MCL, HL, or T-cell lymphoma. First-generation mTOR inhibitors, including rapamycin (sirolimus) and its analogues (everolimus, temsirolimus, and ridaforolimus) have been demonstrated to exert an antitumor activity in vitro against a variety of lymphoma cell lines and primary samples from patients (7, 9, 48, 87). These drugs also synergize in vitro with several agents used to treat lymphoma, including RTX, vincristine, doxorubicin, and bortezomib. Clinical studies of temsirolimus in patients with relapsed or refractory MCL have consistently demonstrated single-agent antitumor activity. This drug is authorized in Europe for treatment of patients with this aggressive B-cell AUTOPHAGY IN LYMPHOPROLIFERATIVE DISORDERS

lymphoma (79). A phase II clinical trial of everolimus in patients with relapsed or refractory lymphoma shows that this drug, when used as a single agent, has some efficacy (ClinicalTrials.gov ID: NCT00436618). However, drug response is observed only in a portion of patients, and durability is limited. Several resistance mechanisms have been hypothesized for this variability, including paradoxical Akt activation (49). Of interest, the prevention of Akt rephosphorylation on everolimus treatment by means of a selective Akt inhibitor greatly enhances everolimus activity in MCL cell lines and primary samples. Furthermore, MCL cells with low response to this combination showed high levels of autophagy. Accordingly, selective knockdown of autophagy genes (i.e., Atg7, Atg5, and Atg3) or pretreatment with the autophagy inhibitor hydroxychloroquine, efficiently overcomes the resistance to Akt/ mTOR inhibitors, leading to the activation of the mitochondrial apoptotic pathway, thus suggesting that counteracting autophagy may represent an attractive strategy for sensitizing MCL cells to everolimus-based therapy (88). Phase I/II clinical trials of everolimus in 531

combination with a variety of agents, including antibodies, multityrosine kinase inhibitors, and combination chemotherapy in patients with relapsed or refractory or newly diagnosed lymphomas are currently underway (ClinicalTrials.gov ID: NCT00869999; NCT01334502; NCT01198665; NCT01665768; NCT01854606; NCT01088048; NCT00474929; NCT00869999; NCT01075321; NCT01567475; NCT00962507; NCT00352443; NCT01453504; NCT00935792; and NCT01341834). Phase I/II clinical trials of ridaforolimus, used as a single agent (ClinicalTrials.gov identifiers: NCT00060632, NCT00060645, and NCT00086125) or in combination with the histone deacetylase (HDAC) inhibitor vorinostat (ClinicalTrials.gov ID: NCT01169532), in patients with relapsed or refractory lymphoma, are also ongoing. Currently, small molecules targeting the kinase domain active site of mTOR, which inhibit both mTORC1 and mTORC2, have been developed. In addition, because of the structural similarity between the mTOR active site and the catalytic subunit of the PI3K isoforms, some of these mTOR kinase inhibitors also induce PI3K inhibition (38). Preclinical studies performed with these second-generation mTOR inhibitors showed increased antitumor activity (89, 90). A phase I/II clinical trial is ongoing to assess the safety and action of the dual mTOR inhibitor CC-223 in lymphoma malignancies (ClinicalTrials.gov ID: NCT01177397). HDAC and multikinase inhibitors In addition to PI3K/Akt/mTOR inhibitors, chemically different classes of drugs capable of targeting multiple pathways involved in lymphomagenesis or the same pathway but at different levels have been tested in preclinical and clinical studies. For example, HDAC inhibitors, modulating both chromatin structure through histone acetylation and the activity of several nonhistone substrates, determine changes in gene transcription and induce a plethora of biological effects ranging from cell death induction to differentiation, angiogenesis inhibition, and modulation of immune responses. Accordingly, vorinostat, a member of this class of drugs, increases both caspase-dependent apoptosis and caspase-independent autophagic cell death when apoptosis is blocked (91). Vorinostat also triggers autophagy through down-regulation of Akt/mTOR signaling and induction of endoplasmic reticulum stress. Moreover, vorinostat treatment upregulates the expression of Beclin-1 and Atg7 and promotes formation of the Atg5–Atg12 conjugate (92). Similarly, the HDAC inhibitors LAQ824 (dacinostat) and LBH589 (panobinostat) cause autophagic cell death in lymphoma cell lines and mouse xenografts when apoptosis is inhibited (93). Overall, these data suggest that although HDAC inhibitors require caspase activity to induce cell death, their therapeutic effect may be preserved, even in the absence of functional apoptosis, by inducing autophagic cell death. However, phase II clinical trials in 532

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patients with relapsed or refractory HL showed that these HDAC inhibitors have limited single-agent activity. Further studies in combination with other active agents are warranted (80). For instance, phase I/II studies evaluating the combination of panobinostat and everolimus in patients with relapsed or refractory lymphoma are ongoing (ClinicalTrials.gov identifiers: NCT00962507, NCT00918333, and NCT00978432). The multikinase inhibitor sorafenib, which inhibits mTOR signaling, when used as a single agent in patients with relapsed or refractory lymphoma, shows modest antitumor activity (74). Of note, sorafenib and the HDAC inhibitor vorinostat interact in a highly synergistic manner to kill, at least in vitro, tumor cells through toxic autophagy (94), providing the rationale for the development of combination regimens. CQ and hydroxychloroquine The multifaceted nature of autophagy and its diverse crosstalk with other programs of cell death is a matter of concern when targeting the autophagic system. As mentioned, tumor cells may use autophagy to survive metabolic stress encountered during chemoradiotherapy, and autophagy inhibitors therefore could enhance the cytotoxicity of cancer treatments. Many clinical trials using CQ and its analogue hydroxychloroquine are being applied to multiple cancer types, including solid tumors and multiple myeloma, in combination with various conventional therapies (95). However, at present, no clinical trials are under way, to test the efficacy of these compounds in lymphoid malignancies.

CONCLUSIONS Despite the crucial role that autophagy plays in tumor initiation and progression, the current scenario emerging from studies targeting autophagy for therapeutic purposes is still puzzling and, by far, contains more questions than answers. An improved understanding of the mechanisms by which autophagy is involved in cancer pathogenesis may lead to the identification of new targets for innovative therapeutic approaches. Drug screening for agonists or antagonists of autophagic activity, including upstream regulators and downstream targets of autophagy, may yield new molecules with therapeutic potential for anticancer therapy. Autophagy inhibitors, such as CQ, could potentiate tumor cell death induced by standard chemotherapy or targeted therapy (e.g., kinase inhibitors) in patients with relapsed or refractory lymphoma. Since kinase inhibitors induce autophagy, the exact therapeutic activity and mechanism of action of autophagy modulators, as determined by ongoing clinical trials in lymphoproliferative disorders, will probably remain controversial, and further preclinical and clinical studies are needed to dissect exactly the level of interactions between autophagy modulators, targeted therapies, and conventional chemotherapy.


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A further key aspect of the autophagic process deals with a diagnostic and prognostic possibility: the development and validation of new autophagy-related biomarkers could lead to the optimization of targeted agents and provide evidence of their activity. Finally, recent gender-based cytopathology studies suggesting a role for sex hormones in modulating autophagy and a different propensity of cells from males and females to autophagy (96, 97) could help to develop specifically tailored treatments improving therapeutic appropriateness.

19.

20.

21. 22. 23.

The authors apologize to all colleagues whose work may not have been cited for space reasons. This work was supported in part by grants from the Italian Ministry of Health (Ricerca Finalizzata 2010) and the Italian Association for Cancer Research (9998 and 11505).

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