The PTEN tumor suppressor gene and its role in lymphoma pathogenesis.

The phosphatase and tensin homolog gene PTEN is one of the most frequently mutated tumor suppressor genes in human cancer. Loss of PTEN function occurs in a variety of human cancers via its mutation, deletion, transcriptional silencing, or protein instability. PTEN deficiency in cancer has been associated with advanced disease, chemotherapy resistance, and poor survival. Impaired PTEN function, which antagonizes phosphoinositide 3-kinase (PI3K) signaling, causes the accumulation of phosphatidylinositol (3,4,5)-triphosphate and thereby the suppression of downstream components of the PI3K pathway, including the protein kinase B and mammalian target of rapamycin kinases. In addition to having lipid phosphorylation activity, PTEN has critical roles in the regulation of genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration. Although PTEN deficiency in solid tumors has been studied extensively, rare studies have investigated PTEN alteration in lymphoid malignancies. However, genomic or epigenomic aberrations of PTEN and dysregulated signaling are likely critical in lymphoma pathogenesis and progression. This review provides updated summary on the role of PTEN deficiency in human cancers, specifically in lymphoid malignancies; the molecular mechanisms of PTEN regulation; and the distinct functions of nuclear PTEN. Therapeutic strategies for rescuing PTEN deficiency in human cancers are proposed.

intrinsic lipid phosphatase activity that negatively regulates the cytoplasmic PI3K/AKT pathway, whereas in the nucleus, PTEN has AKT-independent growth activities. The continued elucidation of the roles of nuclear PTEN will help uncover the various functions of this essential tumor suppressor gene.
In this review, we describe the molecular basis of PTEN loss, discuss the regulation of PTEN expression in lymphoid malignancies, and summarize potential therapeutic targets in PTEN-deficient cancers.

STRUCTURE AND FUNCTION OF PTEN PTEN structure
PTEN is a tumor suppressor gene located on chromosome 10q23.31 that encodes for a 403-amino acid protein that has both lipid and protein phosphatase activities. PTEN gene and protein structures are shown in Figure 1. The PTEN protein contains a sequence motif that is highly conserved in members of the protein tyrosine phosphatase family. Structurally, the PTEN protein is composed of two major functional domains (a phosphatase domain and a C2 domain) and three structural regions (a short N-terminal phosphatidylinositol [4,5]-bisphosphate [PIP 2 ]-binding domain, a Cterminal tail containing proline-glutamic acid-serinethreonine sequences, and a PDZ-interaction motif) [11]. The PIP 2 -binding site and adjacent cytoplasmic localization signal are located at the protein's Nterminal [12,13].

The PI3K/PTEN/AKT/mTOR pathway
PTEN's tumor-suppressing function largely relies on the protein's phosphatase activity and subsequent antagonism of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. Following PTEN loss, excessive PIP 3 at the plasma membrane recruits and activates a subset of pleckstrin homology domaincontaining proteins to the cell membrane. These proteins an N-terminal PIP 2 -binding domain (PBD), a phosphatase domain, a C2 domain, a C-terminal tail containing proline-glutamic acid-serine-threonine sequences, and a PDZ interacting motif at the end. *Mutations on the phosphatase domain that disrupt PTEN's phosphatase activity include the C124S mutation, which abrogates both the lipid and protein phosphatase activity of PTEN, and the G129E mutation, which abrogates only the lipid phosphatase activity of PTEN. The C-terminal tail residues phosphorylated by glycogen synthase kinase 3β (GSK3β) and casein kinase 2 (CK2) are shown. Mutations of S380, T382, and T383 (referred to as the STT) can destabilize PTEN and increase its phosphatase activity. The PIP 2 -binding site and adjacent cytoplasmic localization signal are located at the N-terminal. The N-terminal poly-basic region appears to selectively interact with PIP 2 and contribute to the nuclear accumulation of PTEN. Ubiquitination of PTEN has also been found on K13 and K289.
include phosphoinositide-dependent kinase-1 and AKT family members [14,15]. AKT activation also leads to the activation of the mTOR kinase complex 1 through the inhibition of the phosphorylation of tuberous sclerosis complex tumor suppressors and consequent activation of the small GTPase rat sarcoma (RAS) homologue enriched in brain. The active mTOR complex 1 phosphorylates the p70 ribosomal protein S6 kinase (S6K) and inhibits 4E-binding protein 1 to activate protein translation [16]. Accordingly, the PTEN/PI3K/AKT/mTOR pathway is emerging as a vital target for anti-cancer agents, especially in tumors with mTOR pathway activation.

AKT-independent roles of PTEN
Although AKT pathway activation can explain many of the phenotypes associated with PTEN inactivation, PTEN gene targeting and genetic activation of AKT do not have completely overlapping biological consequences. Using transcriptional profiling, Vivanco et al. identified a new PTEN-regulated pathway, the Jun-N-terminal kinase (JNK) pathway, which was constitutively activated upon PTEN knockdown [17]. In the study, PTEN null cells had higher JNK activity than PTEN positive cells did, and genetic analysis indicated that JNK functioned parallel to and independently of AKT. Thus, the blockade of PI3K signaling may shift the survival signal to the AKTindependent PTEN-regulated pathway, implicated JNK and AKT as complementary signals in PIP 3 -driven tumorigenesis and suggest that JNK may be a therapeutic target in PTEN null tumors.
In addition to its lipid phosphatase function, PTEN also has lipid phosphatase-independent roles. PTEN has been shown to inhibit cell migration through its C2 domain, independent of PTEN's lipid phosphatase activity [18]. In breast cancer, PTEN deficiency has been shown to activate, in a manner dependent on its protein phosphatase activity, the SRC proto-oncogene, non-receptor tyrosine kinase (SRC), thereby conferring resistance to human epidermal growth factor receptor 2 inhibition [19]. Furthermore, PTEN has been shown to directly bind to tumor protein 53 (p53), regulate its stability, and increase its transcription, thereby increasing P53 protein levels [20].

Genetic alteration of PTEN
PTEN loss of function occurs in a wide spectrum of human cancers through various genetic alterations that include point mutations (missense and nonsense mutations), large chromosomal deletions (homozygous/ heterozygous deletions, frameshift deletions, in-frame deletions, and truncations), and epigenetic mechanisms (e.g., hypermethylation of the PTEN promoter region) [21]. Somatic mutations are the main drivers of PTEN inactivation in human cancers, and have been reviewed extensively [22].
PTEN's tumor suppressor function is usually abrogated following mutations in its phosphatase domain, which is encoded by exon 5 [23] (Figure 1). These mutations typically include a C124S mutation that abrogates both lipid and protein phosphatase activity and a G129E mutation that abrogates lipid phosphatase but not protein phosphatase activity [24]. Although the Nterminal phosphatase domain is principally responsible for PTEN's physiological activity, approximately 40% of tumorigenic PTEN mutations occur in the C-terminal C2 domain (corresponding to exons 6, 7, and 8) and in the tail sequence (corresponding to exon 9), which encode for tyrosine kinase phosphorylation sites. This suggests that the C-terminal sequence is critical for maintaining PTEN function and protein stability [21,23,25,26]. However, many tumor-derived PTEN mutants retain partial or complete catalytic function, suggesting that alterative mechanisms can lead to PTEN inactivation.

Transcriptional regulation
In addition to gene mutations, complete or partial loss of PTEN protein expression may impact PTEN's tumor suppression ability. The regulation of PTEN's functions and signaling pathway is shown in Figure 2. Positive regulators of PTEN gene expression include early growth response protein 1, peroxisome proliferatoractivated receptor γ (PPARγ) and P53, which have been shown to directly bind to the PTEN promoter region [27][28][29]. Early growth response protein 1, which regulates PTEN expression during the initial steps of apoptosis, has been shown to directly upregulate the expression of PTEN in non-small cell lung cancer. PPARγ is a ligand-activated transcription factor with anti-inflammatory and anti-tumor effects. The activation of its selective ligand, rosiglitazone, leads to the binding of PPARγ at two PTEN promoter sites, PPAR response element 1 and PPAR response element 2, thus upregulating PTEN and inhibiting PI3K activity. Negative regulators of PTEN gene expression include mitogen-activated protein kinase kinase-4, transforming growth factor beta (TGF-β), nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), IGF-1, the transcriptional cofactor c-Jun protooncogene, and the B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) proto-oncogene, which have been shown to suppress PTEN expression in www.impactaging.com several cancer models [30][31][32]. Research found that IGF-1 could affect cell proliferation and invasion by suppressing PTEN's phosphorylation. In pancreatic cancers, TGF-β significantly suppresses PTEN protein levels concomitant with the activation of AKT through transcriptional reduction of PTEN mRNA-induced growth promotion. c-Jun negatively regulates the expression of PTEN by binding to the activator protein 1 site of the PTEN promoter, resulting in the concomitant activation of the AKT pathway. PTEN transcription is also directly repressed by the leukemiaassociated factor ecotropic virus integration site 1 protein in the hematopoietic system [33].
Intriguingly, recent studies reported a complex crosstalk between PTEN and other pathways. For example, RAS has been found to mediate the suppression of PTEN through a TGF-β dependent mechanism in pancreatic cancer [34], and the mitogen-associated protein kinase/extracellular signal-related kinase pathway has been found to suppress PTEN transcription through c-Jun [35]. Finally, the stress kinase pathways including mitogen-activated protein kinase kinase kinase 4 and JNK promote resistance to apoptosis by suppressing PTEN transcription via direct binding of NF-κB to the PTEN promoter [36]. These findings suggest that the pathways that are negatively regulated by PTEN can in turn regulate PTEN transcription, indicating a potential feedback loop. Studies have also shown that CpG islands hypermethylated in the PTEN promoter lead to the silencing of PTEN transcription in human cancer [37].  Table 1.
Post-translational modifications, such as active site phosphorylation, ubiquitination, oxidation, and acetylation, can also regulate PTEN activity [39]. In its inactivated state, PTEN is phosphorylated on a cluster of serine and threonine residues located on its Cterminal tail, leading to a "closed" PTEN state in which PTEN protein stability is maintained. As PTEN is being activated, dephosphorylation of its C-terminal tail opens its phosphatase domain, thereby increasing PTEN acti-vity ( Figure 2). The phosphorylation of PTEN at specific residues of the C-terminal tail (Thr366, Ser370, Ser380, Thr382, Thr383, and Ser385) is associated with increased protein stability, whereas phosphorylation at other sites may decrease protein stability. Although S370 and S385 have been identified as the major sites for PTEN phosphorylation, mutations of these residues have minimal effects on PTEN function, whereas mutations of S380, T382, and T383 can destabilize PTEN and increase its phosphatase activity, thereby enhancing PTEN's interaction with binding partners [40]. The "open" state of PTEN is more susceptible to ubiquitin-mediated proteasome degradation [13]. One recently identified E3 ligase of PTEN is neural precursor cell-expressed, developmentally downregulated 4, E3 ubiquitin protein ligase 1 (NEDD4-1), which mediates PTEN mono-and poly-ubiquitination [41] (Figure 3). In cancer, the inhibition of NEDD4-1, whose expression has been found to be inversely correlated with PTEN levels in bladder cancer, may  Figure 2).
Acetylation and oxidation also contribute to PTEN activity regulation. PTEN's interaction with nuclear histone acetyltransferase-associated p300/cAMP response element-binding protein (CREB)-binding protein (CBP)-associated factor can promote PTEN acetylation, and this acetylation negatively regulates the catalytic activity of PTEN [49]. Studies have shown that the PTEN protein becomes oxidized in response to the endogenous generation of the reactive oxygen species (ROS) stimulated by growth factors and insulin, and this oxidation correlates with a ROS-dependent activation of downstream AKT phosphorylation [50,51]. Other studies have shown that the PIP 3 -dependent Rac exchange factor 2 and SHANK-associated RH domain interactor proteins bind directly to PTEN to inhibit its lipid phosphatase activity [52, 53]. High P53 expression triggers proteasome degradation of the PTEN protein [54]. In addition to antagonizing the AKT-mouse double minute 2 homolog pathway in a phosphatase-dependent manner, PTEN also can interact with P53 directly in a phosphatase-independent manner, thereby stabilizing P53 [55, 56].

PTEN IN THE NUCLEUS
Growing evidence suggests that the translocation of PTEN from the nucleus to the cytoplasm leads to malignancy. In the nucleus, PTEN has important tumor-suppressive functions, and the absence of nuclear PTEN is associated with aggressive disease in multiple cancers [57-59], implying that nuclear PTEN is a useful prognostic indicator. PTEN is predominantly localized to the nucleus in primary, differentiated, and resting cells, and nuclear PTEN is markedly reduced in rapidly cycling cancer cells [60, 61], which suggests that PTEN localization is related to cell differentiation status and cell cycle stage. High expression levels of nuclear PTEN have been associated with cell-cycle arrest at the G0/G1 phase, indicating a role of nuclear PTEN in cell growth inhibition [62]. PTEN's cytoplasmic and nuclear functions are shown in Figure 3.  (Figure 3). Thus, PTEN is preferentially expressed in the cytoplasm of tumor cells in which PI3K signaling is frequently activated. Nuclear PTEN has an essential role in the maintenance of chromosomal stability. First, PTEN directly interacts with centromere protein C in a phosphatase-independent manner. Second, PTEN transcriptionally regulates DNA repair by upregulating RAD51 recombinase in a phosphatase-dependent manner [68] (Figure 3). The disruption of nuclear PTEN results in centromere breakage and massive chromosomal aberrations. Nuclear PTEN may also play an important part in transcription regulation by negatively modulating the transcriptional activity of the androgen receptor, hepatocyte growth factor receptor, NF-κB, CREB, and activator protein 1. Moreover, nuclear PTEN has been shown to promote p300/CREB-binding proteinmediated p53 acetylation in the response to DNA damage [69,70].
Most of the functions of nuclear PTEN are independent of its phosphatase activity and do not involve the PI3K/AKT pathway. Not only PTEN but also activated PI3K and functional PIP 3 have been detected in the nucleus [71], indicating that nuclear PI3K signaling mediates PTEN's antiapoptotic effect through nuclear PIP 3 and nuclear AKT. Nevertheless, only limited evidence suggests that nuclear PTEN has lipid phosphatase functions, as the nuclear pool of PIP 3 is insensitive to PTEN [72]. www.impactaging.com

PTEN deficiency in T-cell acute lymphoblastic leukemia
PI3K signaling are frequently activated in T-cell acute lymphoblastic leukemia (T-ALL), which mainly due to the absent of PTEN function. Studies have shown that PTEN inactivation plays a prominent role in human T-ALL cell lines and primary patients [73][74][75][76]. Moreover, PTEN mutations have been shown induced resistance to γ-secretase inhibitors, which derepress the constitutively activated NOTCH1 signaling in T-ALL [77]. However, the PTEN mutations detected in these studies vary widely. Gutierrez et al. reported that T-ALL patients had a PTEN mutation rate of 27% and a PTEN deletion rate of 9%, whereas Gedman et al. reported that 27 of 43 (63%) pediatric T-ALL specimens had PTEN mutations. In the latter study, the high frequency of PTEN mutations may have been due to the fact that approximately 50% of the specimens were patients with relapsed disease. Interestingly, all mutations were identified in the C2 domain of PTEN [75,76], not in the phosphatase domain as has been reported for other solid tumors [78].

PTEN deficiency in diffuse large B-cell lymphoma
Published reports of PTEN gene alterations in lymphoid malignancies are summarized in Table 2. Studies have receptors that include receptor tyrosine kinases, G protein-coupled receptors, cytokine receptors, and integrins. PI3K activation converts PIP 2 to PIP 3 , thereby leading to AKT activation, which enhances cell growth, proliferation, and survival. PTEN dephosphorylates PIP 3 and consequently suppresses the PI3K pathway. NEDD4-1 is an E3 ligase of PTEN that mediates PTEN ubiquitination. Polyubiquitination of PTEN leads to its degradation in the plasma, whereas monoubiquitination of PTEN increases its nuclear localization. PTEN can translocate into the nucleus through various mechanisms, including passive diffusion, Ran-or major vault protein-mediated import, and a monoubiquitination-driven mechanism. In the nucleus, PTEN promotes p300-mediated P53 acetylation in response to DNA damage to control cellular proliferation. Nuclear PTEN is also involved in maintaining genomic integrity by binding to centromere protein C (CENPC) and in DNA repair by upregulating RAD51 recombinase (RAD51).
Although several studies have identified discrepancies in PTEN deficiency between DLBCL subtypes, few studies have investigated PTEN localization in different subcellular compartments, not to mention the prognostic value such information would have in de novo cases. Fridberg et al. found a trend towards a stronger staining intensity of cytoplasmic and nuclear PTEN in 28 non-GCB-DLBCL patients [59], most importantly, they found that the absence of nuclear PTEN expression was correlated with worse survival. This interesting evidence should be corroborated in a larger number of primary samples in further studies.

PTEN deficiency in other lymphomas
Previous studies of mantle cell lymphoma (MCL) showed that although the disease had no detectable genetic alterations of PTEN, it did have extremely low protein expression of PTEN. To determine whether the PI3K/AKT signaling pathway is involved in the pathogenesis of MCL, Rudelius et al. investigated pAKT and PTEN expression in primary MCL specimens and cell lines. Of the 31 MCL specimens, 6 had markedly decreased PTEN expression; of the 4 MCL cell lines, 3 had complete loss of PTEN expression [86]. The authors found no phosphatidyl inositol 3-kinase catalytic subunit (PIK3CA) mutations in the primary specimens or cell lines, suggesting that loss of PTEN activates the PI3K/AKT pathway in MCL.
Loss of PTEN protein expression has also been reported in 32% of patients with primary cutaneous DLBCL-leg type and 27% of patients with primary cutaneous follicle center lymphomas. Remarkably, both the expression of miR-106a and that of miR-20a were significantly related to PTEN protein loss (P<0.01). Moreover, low PTEN mRNA levels were significantly associated with shorter disease-free survival [87].

PTEN AND SPECIFIC PI3K ISOFORMS
PI3K comprises a regulatory p85 subunit and a catalytic p110 subunit. Of particular interest, Class IA PI3Ks include three p110 isoforms (p110α, p110β, and p110δ), are primarily responsible for phosphorylating PIP 2 . PIK3CA, the gene encoding the p110α isoform is frequently mutated in various human cancers [88]. In one study, 59% of cases with mutant PIK3CA had increased p-AKT levels. Therefore, the constitutive activation of PI3K is another way by which the PTEN pathway can be disturbed in cancer. In their study of 215 DLBCL patients, Abubaker et al. reported that 8% had PIK3CA mutations and 37% had loss of PTEN. Both PIK3CA mutation and loss of PTEN were correlated with poor survival. However, correlation analysis revealed that most of the PIK3CA mutations occurred in cases with PTEN expression (P=0.0146). Accordingly, 17 cases with PIK3CA mutations were screened for PTEN mutations, and none harbored both PIK3CA and PTEN mutations [89]. This suggests that PIK3CA mutation likely functions as an oncogene in DLBCL by contributing to PI3K pathway activation independently of PTEN deficiency.
Both p110α and p110β may generate distinct pools of PIP 3 . In response to stimuli, p110α produces an acute flux of PIP 3 , which is efficiently coupled to AKT phosphorylation. In contrast, p110β has been proposed to generate a basal level of PIP 3 with little effect on AKT phosphorylation [90]. Moreover, cells with AKT phosphorylation induced by PTEN loss were sensitive to a p110β-specific inhibitor but not a p110α inhibitor both in vitro and in vivo [91,92], which suggests that the enhancement of basal PIP 3 drive oncogenesis in the absence of PTEN. Another study indicated that PTENmutant endometrioid endometrial carcinoma cells may not be sufficiently sensitive to the inhibition of p110β alone and that combined targeted agents may be required for effective treatment [93]. This finding may have been due to the fact that mutations of PTEN and PIK3CA frequently coexist in endometrioid endometrial carcinoma. In contrast, cells with wild-type PTEN seem to engage the p110α or p110δ isoforms. Accordingly, clinical trials of isoform-specific inhibitors are warranted. www.impactaging.com

ENGAGEMENT OF THE PI3K PATHWAY IN B-CELL RECEPTOR SIGNALING
The survival of the majority of B-cell malignancies depends on functional B-cell receptor (BCR) signaling. The successful use of a Bruton tyrosine kinase (BTK) inhibitor to target the BCR pathway in DLBCL has yielded profound discoveries regarding the genetic and biochemical basis of BCR signaling. During BCR signaling, the SRC family kinase LYN phosphorylates the transmembrane protein cluster of differentiation 19, which recruits PI3K to the BCR. The transduction of BCR signaling finally results in the activation of the NF-κB, PI3K, mitogen-associated protein kinase, and nuclear factor of activated T cells pathways, which promote the proliferation and survival of normal and malignant B cells.
BCR signaling is directly affected by frequent mutations in CD79A (immunoglobulin α) and CD79B (immunoglobulin β)-mainly CD79B-which occur in approximately 20% of patients with ABC-DLBCL [94]. Tumor cells harboring CD79B mutations have longer and stronger activation of AKT signaling. Moreover, ABC-DLBCL cell lines with mutated CD79B are more sensitive to PI3K inhibition than those with wild-type CD79B are. Thus, CD79B mutations might be responsible for preventing the negative regulation that interferes with PI3K-dependent pro-survival BCR signaling [95].

Figure 4. Actions of therapeutics targeting PTEN deficiency in lymphoid malignancies. PTEN deficiency is
associated with increased sensitivity to PI3K, AKT, and mTOR inhibitors. In addition, because PI3K is involved in BCR signaling activation, BCR pathway inhibitors may also be effective in PTEN-deficient lymphoid malignancies. SRC family kinase inhibitors include dasatinib (which can also inhibit BTK), saracatinib, bosutinib, SU6656, CGP76030, and KX-01. BTK inhibitors include ibrutinib and AVL-292. Sotrastaurin is a PKCβ inhibitor; A20, a MALT1 paracaspase inhibitor; and MLN120B, an IKKβ inhibitor. SYK inhibitors include fostamatinib and PRT062607. Idelalisib is a PI3Kδspecific inhibitor. MK-2206 is an AKT inhibitor. mTOR inhibitors include everolimus and temsirolimus. to NF-κB activity in ABC-DLBCL, whereas in GCB-DLBCL, PI3K pathway activation but not NF-κB activity is required for survival. Briefly, the "chronic" BCR signaling in ABC-DLBCL is characterized by the many pathways involved with the CARD11-mediated activation of NF-κB signaling, whereas the "tonic" BCR signaling in GCB-DLBCL is characterized by the constitutive activation of PI3K in promoting survival [96,97].
Given these findings, the combination of PI3K pathway inhibitors with BCR pathway inhibitors may enhance the treatment response of PTEN-deficient tumors.

PI3K/AKT/mTOR pathway inhibitors
Owing to PI3K's critical roles in human cancers, PI3K targeting is one of the most promising areas of anticancer therapy development. Since the absent of PTEN is concomitant with PI3K signaling activation, inhibitors that targeting this pathway might play a significant role in the treatment of PTEN-deficient tumors. Growing evidence indicates that multiple solid tumor cell lines and several lymphoid malignancy cell lines with PTEN-deficient are hypersensitive to PI3K inhibitors, which are summarized in Tables 3 and Figure 4.
In addition to PI3K pan-inhibition, several isoformselective PI3K inhibitors have been shown to repress the viability of PTEN-deficient tumors. Notably, the p110β-specific PI3K inhibitor AZD6482 (  displayed remarkable antitumor activity in PTEN-null tumors but failed to block the growth of PTEN-wild-type tumors in mouse models [98]. However, another separate study showed that endometrioid endometrial cancer with PTEN mutation were resistant to p110β-selective inhibition, cell lines' viability was decreased only when p110β-selective inhibition was combined with p110αselective inhibition. Recent findings have highlighted that there is a complex interplay between the Class I PI3K isoforms, inhibition of either α or β single isoform might be compensated by reactivation of another isoform at last [99]. Furthermore, it has been proposed that the dual γ/δ inhibitor CAL-130, specifically targeting p110γ and p110δ isoforms in PTEN deleted T-ALL cell lines [100]. By contrast, Lonetti et al. recently indicated that PI3K pan-inhibition developed the highest cytotoxic effects when compared with both selective isoform inhibition and dual p110γ/δ inhibition, in T-ALL cell lines with or without PTEN deletion [101]. Nevertheless, which class of agents among isoform-specific or pan-inhibitors can achieve better efficacy is still controversial. Other target treatments including AKT, mTOR, dual PI3K/AKT and dual PI3K/mTOR inhibitors also show promising antitumor activity in cell line studies, and some of them have been testing under clinical trials [102][103][104][105][106][107][108][109][110][111] (Table 3,  4).