Signalling Pathways Leading to TRAIL Resistance

One fundamental problem of most malignancies, including those of haematological origin, is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of conventional radio-chemotherapy. In recent years novel compounds have been identified to overcome this major hurdle. Among these, TNF-related apoptosis-inducing ligand (TRAIL) generated considerable enthusiasm for its anticancer therapeutic effectiveness, selectively targeted to a wide range of cancer cells without affecting cells derived from normal tissues and organs. A number of preliminary studies sustain the use of TRAIL-Receptors agonistic antibodies (TRAs) instead of rTRAIL (recombinant TRAIL) in the treatment of tumour cells protected from rTRAIL-induced apoptosis by the expression of cell surface decoy receptors. Although the early clinical trials are promising and well tolerated, the efficacy of these novel approaches is restricted to patients with TRAIL-sensitive tumours. In addition, TRAIL-Rs loss or mutations that can often occur in neoplastic diseases can compromise expected therapeutic results. To overcome TRAIL resistance, novel strategies based on the combination of TRAIL with radiochemotherapy, or with proteasome or histone deacetylase or NF-B inhibitors have been developed. In light of this complex background, this chapter will discuss the current knowledge of the signalling pathways leading to TRAIL resistance and promising targeted therapies in the treatment of haematological malignancies.


Introduction
One fundamental problem of most malignancies, including those of haematological origin, is the development of multiple mechanisms of resistance, which progressively reduce or suppress the therapeutic efficacy of conventional radio-chemotherapy.In recent years novel compounds have been identified to overcome this major hurdle.Among these, TNF-related apoptosis-inducing ligand (TRAIL) generated considerable enthusiasm for its anticancer therapeutic effectiveness, selectively targeted to a wide range of cancer cells without affecting cells derived from normal tissues and organs.A number of preliminary studies sustain the use of TRAIL-Receptors agonistic antibodies (TRAs) instead of rTRAIL (recombinant TRAIL) in the treatment of tumour cells protected from rTRAIL-induced apoptosis by the expression of cell surface decoy receptors.Although the early clinical trials are promising and well tolerated, the efficacy of these novel approaches is restricted to patients with TRAIL-sensitive tumours.In addition, TRAIL-Rs loss or mutations that can often occur in neoplastic diseases can compromise expected therapeutic results.To overcome TRAIL resistance, novel strategies based on the combination of TRAIL with radiochemotherapy, or with proteasome or histone deacetylase or NF-B inhibitors have been developed.In light of this complex background, this chapter will discuss the current knowledge of the signalling pathways leading to TRAIL resistance and promising targeted therapies in the treatment of haematological malignancies.

The TRAIL/TRAIL-Rs system as a novel avenue in anticancer treatment
In recent years novel compounds have been identified to overcome emergence of cancer cells resistance to conventional radio-chemotherapy.Among these, TRAIL generated considerable enthusiasm for its anticancer therapeutic effectiveness, selectively targeted to a wide range of cancer cells without affecting cells derived from normal tissues and organs.TRAIL, also known as Apo-2 Ligand (Apo-2L) (Pitti et al., 1996), is a member of the TNF super-family of cytokines including structurally related proteins that play important roles in regulating cell death, immune response and inflammation.The story of TRAIL begins when a new member of the TNF super-family capable of inducing apoptosis was identified and characterized by virtue of its sequence homology to CD95/Fas/Apo1L (FasL) and TNF (Wiley et al., 1995).death domain (FADD) and the apoptosis initiator caspase-8 are recruited to TRAIL-R1 and/or TRAIL-R2 (Sprick et al., 2000).Although initial studies have attributed a central role to caspase-8 in mediating the apoptotic signal of TRAIL, subsequent studies have demonstrated that apoptosis can be triggered independently through TRAIL-R1 or TRAIL-R2 and proteolytic activation of effector caspases either directly by apical caspase-8 or -10 ( Kischkel et al., 2001) and/or indirectly through Apaf-1-mediated activation of caspase-9 (Green, 2000).Similarly to CD95L, the response to TRAIL is cell-type specific and might be characterized by two distinct cell death pathways (LeBlanc & Ashkenazi, 2003).In the type I extrinsic pathway, extrinsic signals lead to the activation of large amounts of caspase-8 and to the rapid cleavage of executioner caspase-3 prior to loss of mitochondria trans-membrane potential.As a consequence, in type I cells (including leukaemia cells) Bcl-2 over-expression blocks the mitochondrial changes associated with cell death but does not prevent apoptosis that occurs upon death receptors activation.Recent studies have suggested that death receptor induction of apoptosis may depend on the degree of receptor aggregation/multimerization, which may, in turn, depend on the concentration of the death ligand, its form (i.e.soluble versus membrane-bound), the relative DR expression on the cell surface and the array of growth factors and cytokines to which the cells are exposed (Abdulghani & El-Deiry, 2010;Mellier et al., 2010).In the type II intrinsic pathway of apoptosis, intrinsic signals, like DNA damage, growth factor withdrawal or cytokine deprivation, affect the function of Bcl-2 family members (Roos & Kaina, 2006).In fact, in type II cells the extrinsic pathway activated by death receptors is ineffective to recruit, at the DISC level, enough caspase-8 to activate effector caspases.However, through homotypic aggregation at the DISC, caspase-8 is stabilized in an active form and released into the cytosol, where it cleaves its target proteins, most notably the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bid (BH3-interacting-domain death agonist) (Kelley & Ashkenazi, 2004), thus connecting the "intrinsic" mitochondrial pathway to the "extrinsic" DR pathway (Sprick & Walczak, 2004).In turn, truncated Bid (tBid) is able to bind antiapoptotic Bcl-2 family members like Bcl-2, Bcl-XL, Bcl-W and A1 allowing the pro-apoptotic Bcl-2 family members Bax and Bak to engage the mitochondria and induce the release of mitochondrial cytochrome c and Smac (second mitochondria-derived activator of caspases)/DIABLO into the cytosol, where these latter factors promote caspase activation.Actually, cytochrome c forms the "apoptosome" complex with the adaptor protein Apaf-1 resulting in the activation of the apoptosis-initiating protease caspase-9, which then stimulates effector caspases (Green, 2000).Instead, Smac/DIABLO binds to inhibitors of apoptosis proteins (IAPs), preventing their negative-regulatory binding to caspase-9 and -3 and then augmenting apoptosis induction (Salvesen & Abrams, 2004).In this scenario, oncogenic mutations affecting molecules involved in the intrinsic mitochondrial pathway might cause resistance emergence in type II cells, while mutations in the DR pathway could confer resistance to DR-dependent apoptosis especially in type I cells (Fig. 1).It has been demonstrated that the expression pattern of the two killer receptors is broad and partly overlapping, suggesting that they may serve as an alternate or "backup" system, allowing the immune system to control aberrant cells even if one of the two receptors had failed (Greil et al., 2003).Although further investigations are needed to assess differences between DR signalling and regulation, some interesting observations have been reported so far.For example, it has been shown that TRAIL-R1 is activated both by the soluble and the membrane-bound form of the ligand (MacFarlane et al., 2005) whereas TRAIL-R2 is activated by cross-linked soluble and membrane-bound TRAIL ligand but not by the soluble non-cross-linked ligand (Wajant et al., 2001).We previously found a selective radiation-induced upregulation of TRAIL-R1 in different cell lines of haematological origin that sensitized cells to TRAIL cytotoxic activity (Di Pietro et al., 2001), whereas other investigators have suggested a key role for TRAIL-R2 in p53-dependent apoptosis in response to DNA damage both in vitro and in vivo (Burns et al., 2001).Similarly, TRAIL-R2 was up-regulated in B-CLL (chronic lymphocytic leukaemia) cells in response to the small molecule Nutlin-3 in a p53-dependent manner (Coll-Mulet et al., 2006).By means of receptor-selective TRAIL mutant ligands, MacFarlane et al. (2005) demonstrated that CLL cells signal to apoptosis primarily through TRAIL-R1, whereas cross-linked agonistic TRAIL-R2 antibodies facilitate signalling via TRAIL-R2 (Natoni et al., 2007).Other authors have shown different responses of DRs in their cytoplasmic domains that may account for the differences in the activation of these receptors (Thomas et al., 2004).These authors postulated that the binding of ligand and agonistic antibody to the extracellular domain exposes the FADD-binding region differently in the cytoplasmic domain of TRAIL-R1 and TRAIL-R2 to enhance caspase-8 binding and cleavage while promoting recruitment of ancillary proteins.In addition, one more recent paper indicates TRAIL-R2 as a mediator of anoikis of colorectal carcinoma cell lines through the preferential recruitment of the "extrinsic" pathway of apoptosis (Laguinge et al., 2008).

TRAIL decoy receptors
Unlike TRAIL death receptors, TRAIL-R3 and TRAIL-R4 have been originally proposed as "decoy" receptors able to inhibit apoptosis by sequestering TRAIL from the death-inducing TRAIL-Rs or by aggregating with TRAIL death receptors upon binding to the trimeric ligand (Ashkenazi & Dixit, 1999).Nevertheless, it has been demonstrated in primary human CD8+ T cells that the inhibition of TRAIL-induced apoptosis by TRAIL-R4 critically depends on its ligand-independent association with TRAIL-R2 via the NH2-terminal preligand assembly domain overlapping the first partial cysteine-rich domain of both receptors (Clancy et al., 2005).In addition, it has been shown that expression of TRAIL-R1 and/or TRAIL-R2 was necessary but not always sufficient to mediate apoptosis, while expression of TRAIL-R3 and/or TRAIL-R4 often did not correlate with normal and tumour cells resistance to TRAIL effects (Di Pietro & Zauli, 2004).In fact, a number of evidences, based on the use of TRAIL-R agonists rather than over-expression models, have pointed at the primary role of intracellular mechanisms in controlling TRAIL resistance in a number of cell types (Griffith & Linch, 1998;Leverkus et al., 2000), thus cutting down the importance of control at decoy receptors level, whose ability to modulate TRAIL-mediated apoptosis is still controversial.In this regard, particularly intriguing is the role of soluble OPG (Corallini et al., 2008;Secchiero & Zauli, 2008).OPG was initially characterized for its ability to inhibit RANKLstimulated osteoclastogenesis (Emery et al., 1998), but more recent studies highlighted the capability of OPG to counteract the pro-apoptotic activity of TRAIL in a variety of neoplastic cell types, at least in vitro (Schaefer et al., 2007).Of note, the interplay between OPG, RANKL and TRAIL is an important issue in bone and bone marrow biology as well as in the physiopathology of haematological malignancies and in particular of multiple myeloma (MM) (Secchiero & Zauli, 2008).

Molecular mechanisms of TRAIL resistance
The response of individual leukaemia cell lines to TRAIL may depend on which of proapoptotic or pro-survival pathways is dominant.In fact, the sensitivity of leukaemia cell lines to TRAIL is highly variable, with some cell lines demonstrating a marked resistance.In this respect, the large majority of primary haematological tumours are TRAIL-resistant, basically due to the activation of anti-apoptotic signalling pathways (such as Akt and NF-B), over-expression of anti-apoptotic proteins (such as FLIP, Bcl-2, X-IAP), reduced expression of TRAIL death receptors or increased expression of decoy receptors (Testa, 2010;Mellier et al., 2010) (Fig. 1).

TRAIL-receptors
Defects in either of the different molecules involved in TRAIL signalling can lead to TRAIL resistance.With regard to the relationship between TRAIL-receptors expression and TRAILresistance, the data reported in the literature are often contradictory (Russo et al., 2010).An interesting characteristic in the family of DRs is that normal cells are TRAIL resistant, but the molecular basis for TRAIL tumour selectivity is still unclear.In fact, as many chemotherapeutic drugs, TRAIL is not universally active against tumour cell lines, especially primary tumour cells, even expressing DRs on their surface.Dysfunctions of TRAIL-R1/R2 due to oncogenic mutations have been found in different tumours and in different cancer patients (breast, lung, head and neck cancer and non-Hodgkin lymphoma) (Lee et al., 1999;2001).In particular, most tumours mutations map the intracellular domain of TRAIL-R2 that binds the adaptor protein FADD (Shin et al., 2001), known for being essential together with caspase-8 for the DISC assembly.It has been reported that posttranslational modifications, such as O-glycosylation, at the receptor level are essential for TRAIL-R1/R2 full functionality (Wagner et al., 2007), since protein glycosylation could enhance ligand-mediated receptor clustering.Thus, the glycosylation status of TRAIL-R1/R2 has been proposed as a marker of TRAIL sensitivity (Russo et al., 2010).TRAIL resistance has also been linked to the presence of decoy receptors, although their physiological role as well as their impact on normal and cancer cells signalling is still poorly understood.It has been demonstrated that under experimental conditions decoy receptors over-expression could sequester TRAIL, decreasing the functional binding to TRAIL-R1/R2 and attenuating the apoptotic signalling, but experiments under physiological conditions are still missing (Russo et al., 2010).Initially, the preferential expression of TRAIL-R3/-R4 mRNA in normal cells, including peripheral blood lymphocytes, spleen and thymus, was related to the absence of TRAIL cytotoxicity in normal cells, but subsequent studies, using specific monoclonal antibodies (MoAbs), demonstrated that TRAIL sensitivity was not correlated with the relative expression of TRAIL death or decoy receptors (Griffith et al., 1999).It is now accepted that the simple expression of a death or decoy receptor is not an essential feature for apoptosis sensitivity.

PI3K/Akt, MAPK, c-FLIP
Another molecular mechanism responsible for TRAIL resistance emergence is considered the constitutive activation of pro-survival pathways, including Akt pathway.Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that acts as a transducer of many functions initiated by the growth factor receptors that activate phosphatidylinositol 3kinase (PI3K).In this respect, Akt and PTEN (phosphatase and tensin homologue deleted on chromosome 10) constitutive phosphorylation has been linked to TRAIL resistance of acute lymphoblastic leukaemia (ALL) cell lines (Didaa et al., 2008) and acute myeloid leukaemia (AML) patient blasts (Martelli et al., 2006).Interestingly, TRAIL itself can paradoxically activate Akt and downstream targets, like CREB/ATF transcription factors, in leukaemia cells sensitive to its cytotoxic action, loosing to some extent its pro-apoptotic effect (Zauli et al., 2005;Caravatta et al., 2008).Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer (Chen et al., 2001) and protects HL60 leukaemia cells from TRAIL-induced apoptosis by activating the transcriptional factor NF-B and up-regulating c-FLIP (cellular FADD-like interleukin-1beta-converting enzyme-inhibitory protein) (Bortul et al., 2003).c-FLIP acts as an important intracellular inhibitor of TRAIL sensitivity and delivers growth signals by activating NF-B and ERK signalling pathways (Almasan & Ashkenazi, 2003).It is structurally related to caspase-8 (but is devoid of enzymatic activity) since it possesses two death domains that facilitate the binding to the death domain of FADD, thereby preventing association of caspase-8 with the DISC.Over-expression of c-FLIP correlates with TRAIL resistance in several types of cancer, especially in type I cells (MacFarlane et al., 2002).Although it is considered as one of the non-apoptotic NF-B target genes, downwards the anti-apoptotic PKB/Akt and MAPK pathways, it has not been confirmed as a molecular switch between life and death in the same cell (Park et al., 2005).More controversial is the TRAIL-R1/R2-induced activation of PKB/Akt and MAP kinases (Falschlehner et al., 2007).The three key enzymes of MAPK pathway, i.e.ERK, p38-MAPK and JNK, have been often associated with the anti-apoptotic function of TRAIL receptors.Paradoxically, pro-apoptotic effects connect TRAIL and MAPK pathways in different cell models (Frese et al., 2003;Jurewicz et al., 2006).The existence of a death domain alternative DISC complex has also been suggested to explain the TRAIL-dependent activation of MAPK pathways.FADD, caspase-8, RIP (receptor-interacting protein) and TRAF2 (TNF receptorassociated factor 2) might form a cytoplasmic complex upon TRAIL stimulation leading to p38 and JNK activation (Lin et al. 2000).In HUVECs (human umbilical vein endothelial cells) (Zauli et al., 2003) and synovial fibroblasts (Morel et al., 2005), TRAIL has a direct effect on cell survival and proliferation stimulating the PI3K-dependent phosphorylation and activation of PKB/Akt kinase without activation of NF-B.Of note, the activation of an ERK-dependent pathway has been linked to TRAIL-induced maturation of erythroid cells (Secchiero et al., 2004a).

NF-B/ IB
A number of studies of different groups of investigators, including ourselves, have outlined the importance of NF-B activation in determining the resistance/susceptibility of target cells to TRAIL cytotoxicity (Ehrhardt et al., 2003;Zauli et al., 2005), possibly by modulating c-FLIP levels (Bortul et al., 2003).Mounting experimental evidences highlight in TRAIL-resistant cells the activation of NF-B following engagement of TRAIL-R1, -R2, or -R4 (Zauli et al. 2005;Henson et al., 2008).TRAIL activation of NF-B is mediated via TRADD (TNF-R1-associated death domain protein), TRAF2 and RIP and occurs independently of caspase-8/-10 activation (Mühlenbeck et al., 1998;MacFarlane, 2003) (Fig. 1).Importantly, the level of NF-B activation has been related to resistance of leukaemia (Ehrhardt et al., 2003) and neuroblastoma cell lines (Yang & Thiele, 2003) to TRAIL cytotoxicity and its aberrant activation has been involved in promoting tumour migration and dissemination.These findings are consistent with the pleiotropic activity of NF-B transcription factors, which are implicated in the control of cell survival and tumorigenesis (Rayet & Gelinas, 1999).Activation and regulation of Rel/NF-B proteins are tightly controlled by IB proteins, which mask the nuclear localization signal (NLS) of NF-B family members, thereby preventing their nuclear translocation (Baeuerle & Baltimore, 1996).In response to many stimuli, such as TNF, lipopolysaccharide (LPS) or interleukin-1 (IL-1), IB kinase (IKK) is activated and can phosphorylate IBs, which, in turn, can be poly-ubiquitinated and rapidly degraded by the proteasome, allowing the release of sequestered NF-B.After its translocation into the nucleus, NF-B is able to activate its target genes, which, depending on the physiological circumstances (Barkett & Gilmore, 1999), can mediate cell survival or apoptosis.It has been reported, for example, that the TRAIL-mediated recruitment of apical caspase-8/-10 was able to induce the simultaneous activation of both effector caspases (-3, -6, -7) and of NF-B pathway in TRAIL-sensitive myeloid leukaemia cells (Secchiero et al., 2002).As a consequence, the TRAIL cytotoxic/cytostatic activity mediated by effector caspases was reduced by the concomitant pro-survival effect exerted by NF-B.Interestingly, NF-B activation was causally linked to the induction of maturation of the surviving leukaemia cells along the monocytic pathway (Secchiero et al., 2002).Moreover, NF-B activation was paralleled by the absence of degradation and by the nuclear translocation of I in Jurkat T leukaemia cell lines sensitive to the cytotoxic action of TRAIL (Zauli et al., 2005), whereas in TRAIL-resistant primary human erythroblasts NF-B activation was concomitant with IB cytoplasmic localization (unpublished observations).The dual function of NF-B, as an inhibitor or activator of apoptosis, would depend on the relative levels of RelA and c-Rel subunits (Chen et al., 2003).In fact, over-expression of RelA or a transcriptionaldeficient mutant of c-Rel inhibits TRAIL-induced apoptosis in mouse embryonic fibroblasts, whereas depletion of RelA increases cytokine-induced apoptosis (Chen et al., 2003).

IAP, Bcl-2, caspases
Among the anti-apoptotic genes up-regulated by NF-B are included cellular inhibitors of apoptosis proteins 1 and 2 (c-IAP1 and c-IAP2), TRAF1 and TRAF2, c-FLIP and Bcl-XL (Wang et al., 1998).Some of these (e.g.survivin, X-IAP, Bcl-2, Bcl-XL) have been shown to be associated with poor prognosis in AML (Tamm et al., 2000;Paydas et al. 2003).Overexpression of Bcl-2, Bcl-XL, or Mcl-1, loss of Bax or Bak function, increased expression of IAPs and reduced release of Smac/DIABLO from the mitochondria to the cytosol are all events resulting in TRAIL resistance in type II cancer cells (Vogler et al., 2008)(Fig.1) .Another important mechanism through which haematological malignancies can escape TRAIL cytotoxicity is inactivation of the intracellular pro-apoptotic pathways (Ashkenazi et al., 2008).This allows malignant cells not only to escape from TRAIL-induced apoptosis, but also to take advantage of the pro-survival signals induced by TRAIL, which paradoxically may act as a survival cytokine (Fig. 1).In this respect, a possible interplay between the Akt and caspase pathways has been already described in several cell systems (Cardone et al., 1998;Jones et al., 2002;Milani et al., 2003).The picture emerging from these studies is that, when survival signals dominate, Akt impairs the activation of the apical caspases, by directly phosphorylating caspase-9 (Cardone et al., 1998) or by inhibiting the recruitment of procaspase-8/-10 to the DISC (Jones et al., 2002).On the other hand, when pro-apoptotic signals prevail, apical caspase-8/-10 activates downstream caspases, which cleave and inactivate Akt as well as other anti-apoptotic molecules (Milani et al., 2003).All these findings underline the complexity of the TRAIL-mediated intracellular signals, which simultaneously activate pro-apoptotic and anti-apoptotic pathways.The fate of individual malignant cells would depend on which of these pathways prevail within the cell.These effects should be carefully evaluated in the individual assessment of eligibility of cancer patients to TRAIL-based therapy.

TRAIL treatment of haematological malignancies
It has been shown that TRAIL induces growth arrest and apoptosis in cancer cells independently of wt p53 function, Bcl-2 and Bcl-XL (Walczak & Krammer, 2000) and MDR gene expression (Snell et al., 1997).Thus, TRAIL may offer an alternative or complementary approach to conventional anticancer therapy.Unlike other members of the TNF superfamily, such as CD95L and TNF that are precluded from use in systemic anticancer therapy due to their severe toxic side effects (Tartaglia and Goeddel, 1992), TRAIL is effective in selectively killing both in vitro and in vivo a vast array of tumour cells from lung, breast, kidney, colon, prostate, thyroid and skin cancers (Walczak & Krammer, 2000;Papenfuss et al., 2008), without causing significant organ toxicity and inflammation in vivo.

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Signalling Pathways Leading to TRAIL Resistance 209 Moreover, TRAIL exerts a variable cytotoxic activity on haematological malignancies (Snell et al., 1997) and synergistically cooperates with: i) chemotherapeutic drugs, such as etoposide, campthotecin-11, doxorubicin, 5-fluorouracil, taxol (Sabatini et al., 2004;Henson et al., 2008); and ii) ionizing radiation (Chinnaiyan et al., 2000;Di Pietro et al., 2001), causing substantial regression or complete ablation of solid (colon and mammary) cancers in animal models.Besides acting as a tumour suppressor in vivo in primary tumours, TRAIL could play a substantial role in suppressing tumour metastasis.In fact, it has been observed that this cytokine may partially limit the formation of hepatic metastases of a variety of mouse tumours (Seki et al., 2003).A study performed in TRAIL -/-null mice demonstrated that the incidence of spontaneous lymphoid malignancies was increased by 25% in comparison with control animals (Zerafa et al., 2005), suggesting a crucial role of TRAIL in the immunesurveillance against lymphoid malignancies.Although it is not established whether TRAIL causes liver toxicity in humans (Jo et al., 2000;Lawrence et al., 2001), pre-clinical studies performed in mice and non-human primates indicated that rTRAIL protein promotes potent apoptosis-inducing activity against tumour cells without a relevant systemic toxicity (Walczak et al., 1999).Phase I and phase II clinical trials in patients with advanced solid tumours or non-Hodgkin lymphoma (NHL) appeared to go in the same direction, indicating that both rTRAIL and TRAs are safe and well tolerated (Koschny et al., 2007;Tolcher et al., 2007).Therefore, TRAIL ligand and TRAs are strong candidates for an effective but tolerable treatment of solid cancers, either used alone or in combination with radio-chemotherapy.

Acute myeloid leukaemia (AML)
The cytotoxic activity of TRAIL has been evaluated in haematological diseases by different groups of investigators, including our research group (Secchiero & Zauli, 2008;Sancilio et al., 2008;Impicciatore et al., 2010).Overall the activity of TRAIL as a single treatment in acute and chronic leukaemia is poor.Unlike the poor outcome of TRAIL treatment in primary AML blasts, continuous cell lines derived from AML display a pronounced sensitivity to the apoptotic action of TRAIL (Snell et al., 1997;Secchiero et al., 2004b).Moreover, when TRAIL is used in combination with chemotherapeutic agents (fludarabine, cytosine arabinoside or daunorubicin) additive or super-additive apoptotic effects are obtained, due to the ability of these agents to activate apical caspase-8/-10 ( Jones et al., 2003).In line with these findings, other authors demonstrated that triterpenoids, natural and synthetic compounds with demonstrated anti-tumour activity, induced a substantial increase in cell death in both B-CLL and AML blasts, by inducing a concentrationdependent decrease in the levels of FLIP protein (Suh et al., 2003;Pedersen et al., 2002).A recent report has related the poor response of AML to the simultaneous expression of death and decoy receptors (Inukai et al., 2006), whereas co-expression of death receptors with the decoy receptor TRAIL-R3 resulted in significant shortened overall survival of AML patients (Chamuleau et al., 2011).Another weak point in leukaemia treatment is represented by p53 gene deletions or mutations, that usually occur in less than 15% of AML cases.To augment the poor response of AML to TRAIL cytotoxicity, Secchiero et al. (2007) have recently adopted the strategy to combine rTRAIL with Nutlin-3, a potent non-genotoxic activator of the p53 pathway (Impicciatore et al., 2010).In this investigation Nutlin-3 synergized with TRAIL in inducing apoptosis both in AML cell lines and primary M4-type and M5-type AML blasts, but not in mut p53 AML cells, suggesting that the combined treatment of Nutlin-3 plus TRAIL might offer a novel therapeutic strategy for wt p53 AML cells.Clodi et al. (2000) demonstrated that TRAIL has a modest activity in primary ALL since it killed a maximum of 29% of precursor-B-cell blasts within 18 hours treatment against the 75% of the sensitive Jurkat cell line.Childhood T-ALL is frequently accompanied by hyperleukocytosis at disease presentation, suggesting that T-ALL tends to acquire mechanisms for escaping immune surveillance of the hosts that promote its rapid clonal expansion.Clinically, dramatic advances have been made in the treatment of childhood T-ALL.However, despite the use of intensive risk-adapted chemotherapy, treatment failure occurs in approximately 25% of patients (Goldberg et al., 2003).Since the prognosis of relapsed T-ALL remains dismal, the development of a new therapeutic modality is urgently required.In a recent report on T-ALL cell lines and primary samples of childhood T-ALL the failure of anti-leukaemic activity of soluble rTRAIL was linked to the low cell surface expression levels of TRAIL-R1 and TRAIL-R2, which could not be modified by the demethylating agent 5-aza2'deoxycytidine (Akahanea et al., 2010).

Chronic myeloid leukaemia (CML)
Only few studies have investigated the effects of rTRAIL on CML blasts.An interesting study of Tanaka et al. (2007) demonstrated increased levels of serum TRAIL and TRAIL mRNA in neutrophils of CML patients during IFN therapy, suggesting a novel antineoplastic role of neutrophils mediated by the expression/release of TRAIL.Since neutrophils, unlike activated lymphocytes, display a low susceptibility to TRAIL cytotoxicity (Meurette et al., 2006), these findings are of particular value.Other studies have shown that TRAIL, used as a single agent, significantly reduces the number of myeloid colonies and clusters from patients affected with CML and myelodysplastic syndromes (MDS) (Zang et al., 2001;Uno et al., 2003), while normal human stem cells treated with high doses of TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice (Zang et al., 2001).Moreover, it was recently demonstrated that the loss of Bcr-Abl in imatinib-resistant CML cells leads to the down-regulation of c-FLIP and the subsequent increase in TRAIL sensitivity, suggesting that TRAIL could be an effective strategy for the treatment of imatinib-resistant CML with loss of Bcr-Abl (Park et al., 2009).

B-type chronic lymphocytic leukaemia (B-CLL)
A pressing need for the identification of novel therapeutic approaches regards B-CLL disease.It is known in fact that B-CLL patients may have initial clinical responses to alkylating agents, such as chlorambucil, or adenosine analogs, such as fludarabine, but they ultimately become refractory to therapy.Preliminary studies, carried out on cell lines and a modest number of primary samples, have shown a low cytotoxic activity of TRAIL on lowgrade B-CLL (MacFarlane et al., 2002).Collectively, low-grade B-cell malignancies constitute one of the most common form of potentially lethal cancer in Europe and North America, with B-CLL representing the most prevalent of these disorders (Reed et al., 2002).B-CLL is characterized by the accumulation of mature non-proliferating B cells defective in apoptotic mechanisms and resistant to anticancer therapy.A number of molecular defects and biologic features have been identified in this pathology.Olsson et al. (2001) revealed a higher constitutive expression of the long form of FLIP (FLIP-L) in B-CLL as compared to normal tonsillar B cells.MacFarlane et al. (2002) demonstrated that resistance to TRAIL was upstream of caspase-8 activation, since little or no caspase-8 was processed in TRAIL-treated www.intechopen.comSignalling Pathways Leading to TRAIL Resistance 211 B-CLL cells.As a consequence, the possibility of sensitizing B-CLL cells to TRAIL-mediated cytotoxicity could reside in the modulation of c-FLIP levels or in the up-regulation of DR surface expression.In consistence with this hypothesis, the combination of TRAIL with anti-CD95 ligand has proved effective in inducing apoptosis of CD40-activated B-CLL cells (Dicker et al., 2005).A more recent study, aimed at evaluating molecular mechanisms of TRAIL resistance of B-CLL, identified a different TRAIL sensitivity of Zap-70low and Zap-70high B-CLL subsets, proposing this negative prognostic marker as responsible to redirect TRAIL signallling from pro-apoptotic to pro-inflammatory pathway (Richardson et al., 2006).

Lymphoma
The potential therapeutic use of TRAIL has been also explored in the therapy of refractory diffuse large B-cell lymphoma (DLBCL) (Cillessen et al., 2006), cutaneous T-cell lymphoma (CTCL) (Braun et al., 2007), mantle B cell lymphoma (MCL) (Roue et al., 2007) and plasmacytoid dendritic cell (PDC) leukaemia (Blum et al., 2006), which shows a particularly aggressive clinical course.In particular, Cillessen et al. (2006) have shown that 12 out of a total of 22 DLBCL samples, including 7 clinically chemotherapy-refractory lymphomas, were sensitive to TRAIL-mediated apoptosis.TRAIL cytotoxic effects were also detected in CD4+CD56+ PDC leukaemia (Blum et al., 2006), as well as in the majority of MCL cell lines and primary cultures investigated by Roue et al. (2007), whose research group used TRAIL in combination with the IB kinase inhibitor BMS-345541 to overcome resistance of MCL samples.In a recent review (Sancilio et al., 2008), we suggested the possibility to combine two biologically active and well-tolerated agents with different mechanisms of action, such as rituximab and agonist MoAbs against DRs, as an attractive treatment strategy for patients affected with B-cell lymphoma.The in vivo mechanisms through which rituximab mediates its effects have not been fully elucidated, though ADCC (antibody-dependent cellular cytotocxicity), CMC (complement-mediated cytotoxicity) and apoptosis have been suggested and supported by several studies (Bonavida, 2007).By contrast, a number of in vitro experimental evidences have been obtained in B-NHL cell lines as a model system (Bonavida, 2007).The findings here described demonstrate that rituximab treatment is able to modulate different signalling pathways, like p38-MAPK, Raf-1/MEK/ERK1/2 and NF-B, leading to the down-regulation of Bcl-2/Bcl-XL gene products, known players of the intrinsic apoptotic pathway.Through this mechanism, chemo-sensitization of drug-resistant B-NHL cell lines to various drug-induced apoptosis could be achieved.

Multiple myeloma (MM)
A number of studies from several groups of investigators have allowed clearly establishing that myeloma is the most susceptible haematological malignancy to rTRAIL used as a single agent (Secchiero et al., 2004b).In particular, Gazitt (1999) demonstrated for the first time that TRAIL induces substantial apoptosis in freshly isolated, flow-sorted myeloma cells obtained from different MM patients.Subsequently, the same group of investigators (Liu et al., 2003) demonstrated that TRAIL is a potent inducer of apoptosis, independent of Bcl-2.Moreover, consistently with the potential role of NF-B and Akt pathways in counteracting apoptosis induction by either chemotherapy or TRAIL, the cell permeable nuclear factor NF-B inhibitor SN50 sensitized TRAIL-resistant MM cells to TRAIL cytotoxicity (Mitsiades et al., 2002) and the Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-Ooctadecylcarbonate-induced cell death of both Dex-and Doxo-sensitive and -resistant cell clones.Interestingly, also thalidomide, which holds great promise as a new anti-neoplastic agent for the treatment of refractory MM, triggers activation of caspase-8 and downregulates NF-B activity and c-FLIP (Mitsiades et al, 2002).These studies form the basis for clinical trials of these agents, alone and coupled with conventional and novel therapies, to improve outcome in MM.It is worth underlining that while the potential therapeutic use of rTRAIL or TRAs in most myeloid and lymphoid malignancies is still to be evaluated, rTRAIL appears to be a very promising candidate for the therapy of MM, either alone or in combination with valproic acid, a histone deacetylase inhibitor, arsenic trioxide, IFN, or with the low-molecular-weight Smac mimetic LBW242 (Secchiero & Zauli, 2008).Moreover, the use of specific anti-TRAIL-R1 or anti-TRAIL-R2 agonistic antibodies, more than the treatment with TRAIL itself (Locklin et al., 2007), has proved an effective strategy to counteract OPG-mediated effects and increase TRAIL-induced apoptosis of MM cells (Secchiero & Zauli, 2008).Of note, other preclinical studies aimed at targeting the RANK/RANKL/OPG pathway have paved the way to clinical experimentation likely to lead to new therapeutic approaches (Buckle et al., 2010).

Novel strategies to overcome TRAIL resistance
During the last decades, a better understanding of cancer biology has led to the development of new promising therapeutic approaches, based on "molecular targeted" drugs, directed against specific "target" molecules playing a key role in tumour maintenance (Urruticoechea et al., 2010).Based on the principle that inhibiting as many targets as possible reduces the emergence of drug resistance, the use of combined therapies or multi-target inhibitors is gaining field in the design of new treatments.A number of chemical and physical anticancer strategies have been developed to bypass TRAIL resistance, based on the combination of rTRAIL or agonistic antibodies with chemotherapeutic agents, irradiation, or targeted small molecules, like proteasome, histone deacetylase or NF-B inhibitors (Testa, 2010;Russo et al., 2010).The agents used in combination with TRAIL either enhance TRAIL-R1/-R2 expression or decrease expression of anti-apoptotic proteins (c-FLIP, X-IAP, Bcl-2) (Mellier et al., 2010) (Fig. 2).Many of these combinatorial therapies hold promise for future developments in the treatment of haematological malignancies since they may reduce excessive systemic toxicity toward normal cells and resistance of tumour cells after recurrent treatments.We and other authors have demonstrated that TRAIL-mediated cytotoxicity is increased by ionizing radiation and chemotherapy in both myeloid and erythroid leukaemia cell lines as well as in T lymphoma cell lines (Gong & Almasan, 2000;Di Pietro et al., 2001;Sabatini et al., 2004;Zauli et al., 2005;Caravatta et al., 2008;Impicciatore et al., 2010;Signore et al., 2011).Although an increasing number of drugs warrant further investigation as potential new strategies for the treatment of solid tumours or AML in combination with soluble rTRAIL (Suh et al., 2003), only in few cases the efficacy of the combined treatments has been proved in vivo and a general consensus on how chemotherapy and radiotherapy may synergize with TRAIL therapy is far to be reached (Russo et al., 2010).

TRAIL-death receptor-targeted treatment
A number of receptor-specific TRAIL-variants and agonistic antibodies have been recently developed.Some of these soluble rTRAIL and MoAbs targeting TRAIL-R1 and/or TRAIL-R2 (TRAIL receptor agonists, TRAs) are progressing to phase I/II clinical trials (Mahmood & Shukla, 2010).The clinical use of TRAs is a very promising and innovative approach to increase selectivity and reduce undesired toxicity of cancer treatments in comparison with modern anticancer drugs (protein kinase inhibitors or MoAb agonists for growth receptors) (Russo et al., 2010).These compounds were generated to selectively bind and activate their respective DRs without affecting decoy receptors or OPG.DRs engagement, using recombinant death ligands or agonistic antibodies, leads to the activation of both extrinsic and intrinsic apoptosis pathways, while, generally, chemotherapy or radiotherapy triggers the mitochondrial/intrinsic pathway (Fig. 2).Therefore, the conventional therapeutic approach could be implemented by DR-induced apoptosis when DRs are expressed and functional on tumour cells.As already mentioned, although soluble rTRAIL as well as TRAs are not completely free from toxicity, both reagents elicit a significant lower hepatotoxicity when administered systemically compared to CD95 receptor agonists (Lawrence et al., 2001).Besides the advantage of an improved specificity and a lower toxicity of TRAs over TRAIL ligand, pharmacokinetic studies performed in primates and humans have shown that these agents have a longer half-life (around 15 days) than soluble TRAIL (30 min) that makes them easier to dose and administer (Duiker et al., 2006).Preclinical studies performed in vitro in cultured human cell lines and in vivo in murine xenograft cancer models (Cretney et al., 2007) showed favourable results when TRAs were used as single agents and enhanced cytotoxicity when they were combined with chemotherapy or radiotherapy (Marini et al., 2006).In particular, HGS-ETR1 (anti-TRAIL-R1, mapatumumab) as well as HGS-ETR2 (anti-TRAIL-R2, lexatumumab) was able to induce apoptosis in primary and cultured lymphoma cells increasing cell death when associated either with conventional chemotherapy (doxorubicin) or novel drugs like proteasome inhibitors (bortezomib) (Georgakis et al., 2005).As well, multiple solid tumours including lung, colon and renal carcinoma were found responsive to TRAs treatment used alone or in combination with chemotherapy (Pukac et al., 2005).To date, the fully humanized MoAbs HGS-ETR1, HGS-ETR2 and HGS-TR2J (anti-TRAIL-R2) (all three from Human Genome Sciences, Rockville, MD) are used in ongoing trials for the treatment of advanced solid tumours, lymphoma or MM (Mahmood & Shukla, 2010).A number of excellent reviews on different therapeutic approaches to specifically target TRAIL and DR pathways have been recently published (Ashkenazi et al., 2008;Papenfuss et al., 2008;Mahmood & Shukla, 2010;Russo et al., 2010).Of particular interest is the current use of rTRAIL and TRAs for the treatment of B cell malignancies (Mahmood & Shuka, 2010).As shown by other authors, the DR pathway is intact and functional in various types of cancers, including B-cell lymphomas (Snell et al., 1997;Georgakis et al., 2005).B-cell sensitivity to TRAs is a fundamental requirement for therapeutic efficacy, since TRAIL-R1 and TRAIL-R2 mutations, observed in NHL as well as in other human tumours (Lee et al., 2001), make neoplastic B cells insensitive to TRAIL and, presumably, to agonistic antibodies mimicking its action.TRAIL-R1 and TRAIL-R2 map to human chromosome 8p21-22, a site of frequent allelic loss in tumours.This led to the hypothesis that, as potential tumour suppressors, TRAIL-Rs may also harbour somatic mutations in human tumours.The most frequent mutations identified so far concern TRAIL-R2 and affect the intracellular domain of the receptor, i.e. the FADD-binding domain, and, as a consequence, its capability of inducing apoptosis (Bin et al., 2007).Although still poor is the knowledge of how TRAIL-Rs mutations affect signalling events, it is predictable that a patient displaying a TRAIL-R2 mutation would not benefit from treatment with either rTRAIL or an anti-TRAIL-R2 antibody but from treatment with mapatumumab or a modified version of rTRAIL able to target only one death receptor (MacFarlane et al., 2005).Similarly to TRAIL, TRAs (mapatumumab and lexatumumab) are capable of inducing antilymphoma effects both in vitro and in vivo (Motoki et al., 2005).In particular, it has been recently demonstrated that mapatumumab can trigger apoptosis through caspase-8 activation via the extrinsic apoptotic pathway (Maddiplata et al., 2007).Various groups of investigators have shown that the activation of the TRAIL-Rs by either ligands or MoAbs sensitizes cancer cells to the effects of various chemotherapeutic and/or biological agents (Secchiero et al., 2007), although in a recent report no correlation between the degree of antitumour in vitro activity of mapatumumab and TRAIL-R1 antigen density has been shown (Maddiplata et al., 2007).To explain their results these investigators hypothesized differences in the receptor hetero-dimerization between various B-cell lymphoma cells upon TRAIL-R1 binding to mapatumumab.In fact, it has been published that following in vitro exposure to TRAIL ligand or TRAIL-R1 agonists, other DRs are recruited via trimerization, leading to signal transduction and apoptosis (Cretney et al., 2007).Depending on the type of DR undergoing trimerization upon TRAIL-R1 binding the intensity and type of response could change.In theory, the same principle could justify the absence of anti-tumour activity of lexatumumab despite ample surface expression of TRAIL-R2 in the cell lines tested in vitro (Maddiplata et al., 2007).Further studies are needed to address this issue.

Proteasome and histone deacetylase inhibitors
Inhibition of NF-B (e.g. with mutant forms of IB or proteasome inhibitors) has also been shown to increase TRAIL responsiveness (Sayers & Murphy, 2006).In this respect, besides the clinical use of bortezomib (Velcade, PS-341) for the treatment of multiple myeloma (Sayers & Murphy, 2006), it has been recently reported that treatment with the proteasome inhibitors MG-132 and PS-341 is associated with the up-regulation of TRAIL and its death receptors, TRAIL-R1/TRAIL-R2, in primary B-CLL cells and in the Burkitt lymphoma cell line, BJAB (Kabore et al., 2006).Interestingly, the combined treatment with TRAIL or TRAs and proteasome inhibitors leads to a significant apoptosis induction in B-CLL but not in normal B cells (Kabore et al., 2006).DRs up-regulation by PS-341 was attributed to TRAIL-R2 mRNA stabilization and the consequent increased receptor half-life (Kamdasamy & Kraft, 2008).In addition to the proteasome inhibitors, inhibition of histone deacetylase (HDAC) class I sensitizes B-CLL to TRAIL-induced apoptosis (Hamilton et al., 2010).An aberrant regulation of gene expression due to alterations in histone acetyltransferase (HAT) or HDAC recruitment and activity has been constantly found in both solid and haematological tumours (Mai & Altucci, 2009).Therefore HDAC can be considered as potential therapeutic targets of human malignancies.Interestingly, the reduction of TRAIL protein degradation has been recently observed in thyroid cancer cells and proposed as a novel action of HDAC inhibitors (Borbone et al., 2010).It is worth outlining that HDAC inhibitors exert anti-tumour effects at doses that are well tolerated by the patients.Hydroxamic acids, such as SAHA (Vorinostat, Zolinza), were recently approved by the US FDA for the treatment of cutaneous manifestations in patients affected with advanced refractory CTCL.In fact, similarly to what has been seen in B-CLL, CTCL cell lines show pronounced resistance to TRAIL cytotoxicity (Braun et al., 2007).Lastly, a phase I study, currently recruiting participants, will use vorinostat in combination with cytarabine and etoposide for the treatment of patients with relapsed and/or refractory acute leukaemia, MDS or myeloproliferative disorders (see for details http://clinicaltrials.gov).

Small molecules and natural compounds
Besides oncogenes over-expression and cell cycle control mechanisms disruption, mutations in apoptotic regulators (namely p53) are very frequent in cancer cells and represent for them a way to escape toxic effects inducible with radio-chemotherapy.As an alternative strategy to restoring transcriptional activation to mutant p53 proteins in solid tumours, small molecule selective inhibitors of p53/MDM2 interaction (Nutlins) are emerging as an innovative tool in the treatment of malignancies expressing wt p53 including haematological disorders (Secchiero et al., 2008;Impicciatore et al., 2010).Nutlins were the first potent and selective small molecules, antagonists of the p53/MDM2 interaction, to be identified 7 years ago (Vassilev et al., 2004).Since then several classes of small-molecule inhibitors with distinct chemical structure have been reported (Shangary & Wang, 2009), although only Nutlin-3 has been extensively evaluated for its therapeutic potential and mechanism of action in human cancer and represents a promising therapeutic candidate for drug development (Shangary & Wang, 2009).Several authors have investigated the effects of Nutlins, used alone or in combination with other therapeutic agents, on primary cells, different cell lines and tumour xenografts (Kojima et al., 2005;Lehmann et al., 2007).In particular, it has been reported that the active enantiomer Nutlin-3a induces i) increased levels of p53, ii) p53-and p21-dependent cell cycle arrest and iii) p53-dependent apoptosis in a number of solid tumours and haematological malignancies including primary AML (Kojima et al., 2005), MM (Stuhmer et al., 2005), B-CLL (Coll-Mulet et al., 2006) and Hodgkin lymphomas (HL) (Drakos et al., 2007).Unlike radiation and conventional chemotherapy, MDM2 inhibitors induce accumulation and activation of p53 in cancer and normal cells without inducing DNA damage or post-translational modifications of p53.Nutlins in fact restore p53 function in wt p53 tumour cells without inducing p53 phosphorylation and with limited effects on primary cells (Vassilev et al., 2004).Interestingly, when used at concentrations higher than 10 mM, Nutlin-3, MI-63 and MI-219 are able to inhibit cell proliferation even in cancer cells lacking wt p53 (Shangary & Wang, 2009).In response to Nutlin-3 treatment TRAIL-R2 is up-regulated in B-CLL cells in a p53-dependent manner (Coll-Mulet et al., 2006).Moreover, Nutlins reduce the MDM2 ability to stimulate p53 degradation and represent a promising approach for improving radiotherapy effects especially for tumours over-expressing MDM2 such as sarcomas, solid tumours (Momand et al., 1998) and NHL (Finnegan et al., 1994).Besides the use of a broad range of protein inhibitors, chemotherapeutic agents or irradiation to exert synergistic effects with TRAIL action (Mahalingam et al., 2009), more recently the use of natural compounds, including polyphenols, has gained increasing interest due to their relative safety and anti-tumour efficacy in preclinical models (Jacquemin et al., 2010).Actually, it has been demonstrated that a number of natural compounds are able to enhance TRAIL-induced apoptosis in leukaemia cells (Fas et al., 2006;Russo et al., 2007;Hussain et al., 2008;Sung et al. 2010).In particular, it has been shown that wogonin, derived from a popular Chinese herb, attenuates NF-B activity (Fas et al., 2006), whereas curcumin, responsible for the yellow colour of the spice turmeric, upregulates TRAIL-R2 expression and inactivates NF-B in a ROS-dependent manner in a number of solid tumours including Burkitt's lymphoma (Hussain et al., 2008).Moreover, Russo et al. (2007) reported that leukaemia cell lines were efficiently sensitized by quercetin and TRAIL co-treatment through the inhibition of the Akt pathway, while Sung et al., 2010 observed other survival proteins down-regulation after TRAIL and triterpenoids co-treatment.Taken together, these findings demonstrate that nongenotoxic natural molecules or small compounds enhance TRAIL-mediated killing of tumour cells with reduced side effects compared to conventional radio-chemotherapy.

Conclusion
A number of preliminary studies sustain the use of TRAs instead of rTRAIL in the treatment of tumour cells protected from TRAIL-induced apoptosis by the expression of cell surface decoy receptors.Although the early clinical trials are promising and well tolerated, it is worth outlining that the utility of both rTRAIL and agonistic anti-TRAIL-Rs antibodies therapies is restricted to patients with TRAIL-sensitive tumours.To restore TRAIL sensitivity in cancer cells novel compounds have been identified and are currently used in combined protocols with TRAIL ligand/TRAs or conventional radio-chemotherapy.Once mechanisms of action/resistance to TRAIL signalling are better understood, approaches to predict patient response and optimize combination regimens may be developed to overcome primary and acquired resistance on the trail to a personalized treatment of cancer.

Fig. 1 .
Fig. 1.Schematic representation of key mechanisms involved in TRAIL resistance of haematological malignancies.Activation of TRAIL-Rs can trigger both death and survival pathways, depending on the cell system and environmental conditions.TRAIL-R1 and TRAIL-R2 can lead to apoptotic cell death through the recruitment of FADD and the following cleavage of caspase-8 and -10.Both DRs together with the "decoy" TRAIL-R4 are also involved in the priming of survival genes through the activation of a) NF-B and JNK pathways triggered by the engagement of TRAF2 and RIP; b) PI3K/Akt and MAPK/ERK1/2 pathways, by means of still unclear mechanisms (highlighted with a question mark).Other mechanisms leading to TRAIL resistance include different caspase or PI3K physiological inhibitors.

Fig. 2 .
Fig. 2. Schematic representation of molecular mechanisms of novel strategies aimed at restoring TRAIL sensitivity of haematological malignancies.RAD: radiotherapy; CHT: chemotherapy.See text for other abbreviations.