Accepted Manuscript Role of tlrs in multiple myeloma and recent advances Krishan K. Thakur , Nityanand B. Bolshette , Cristiana Trandafir , Vinayak S. Jamdade , Alexandru Istrate , Ranadeep Gogoi , Andrei Cucuianu PII:

S0301-472X(14)00753-X

DOI:

10.1016/j.exphem.2014.11.003

Reference:

EXPHEM 3205

To appear in:

Experimental Hematology

Received Date: 20 June 2014 Revised Date:

2 November 2014

Accepted Date: 10 November 2014

Please cite this article as: Thakur KK, Bolshette NB, Trandafir C, Jamdade VS, Istrate A, Gogoi R, Cucuianu A, Role of tlrs in multiple myeloma and recent advances, Experimental Hematology (2014), doi: 10.1016/j.exphem.2014.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract: The role of TLRs in inflammation-induced malignancies, like Multiple Myeloma. TLR- Toll-Like Receptor; TRIF- TIR-Domain-Containing AdapterInducing Interferon-Β; NF-κB - Nuclear Factor- kB; INF- interferon

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ROLE OF TLRs IN MULTIPLE MYELOMA AND RECENT ADVANCES Krishan K. Thakura*, Nityanand B. Bolshettea*#, Cristiana Trandafirb,

a

Laboratory of Biotechnology,

Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER),

b

Faculty of Medicine,

400349, Cluj-Napoca, Romania c

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University of Medicine and Pharmacy ”Iuliu Hațieganu”,

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Gauhati Medical College, Guwahati-781032, Assam, India

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Vinayak S. Jamdadec, Alexandru Istrateb, Ranadeep Gogoia, Andrei Cucuianud

Laboratory of Molecular Pharmacology and Toxicology,

Department of Pharmacology and Toxicology,

National Institute of Pharmaceutical Education and Research (NIPER),

d

Department of Hematology,

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Gauhati Medical College, Guwahati-781032, Assam, India

"Ion Chiricuta" Cancer Institute, Bvd 21 Decembrie Nr 73,

Corresponding Author

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#

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400124, Cluj-Napoca, Romania

Nityanand B Bolshette

Department of Biotechnology,

National Institute of Pharmaceutical Education and Research (NIPER), Gauhati Medical College, Guwahati-781032, Assam, India Ph: +919706798537 Email: [email protected] (* Both the authors, Krishan K. Thakur, Nityanand B. Bolshette contributed equally as a first authors)

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Abstract Multiple Myeloma (MM) is a hematological malignancy characterized as an abnormal proliferation and invasion of plasma cells into the bone marrow. Toll-like receptors (ТLRs) connect the innate and adaptive immune responses and represent a significant and potentially linking element between inflammation and

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cancer. When TLRs bind to their ligands, they trigger two major signaling pathways such that both share overlapping downstream signals: one is a MyD88 dependent production and activation of Nuclear FactorκB (NF-κB), whereas the other is a MyD88 independent production of type-I interferon. While MyD88 pathway results in proinflammatory cytokine production, the other pathway stimulates cell proliferation. Dysregulations of these pathways may eventually lead to abnormal cell proliferation and MM. In spite of

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recent biomedical advances, MM continues to be an incurable disease. There are an increasing number of TLR-based therapeutic approaches, currently tested in a number of pre-clinical and clinical studies. We

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hereby attempt to outline in detail the currently available information on TLRs in various types of cancer.

Keywords: - Multiple Myeloma (MM), Toll-like Receptors (TLRs), Nuclear Factor-κB (NF-κB),

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MyD88, cell proliferation

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Introduction Multiple myeloma (MM) is currently the second most common hematological malignancy after chronic lymphocytic leukemia (CLL), with its incidence increasing lately [1]. The CRAB criteria (elevated

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calcium level, renal failure, anemia and osteolytic bone lesions [2-4] ) clinically define this pathology. In MM, monoclonal plasma cells multiply and secrete large quantities of monoclonal immunoglobulins. Usually, normal immunoglobulins in the bloodstream decrease in number and leave MM patients susceptible to infection or inflammation [5]. This renders therapeutic approaches difficult; MM is practically incurable, as the currently available treatments only lead to transient responses [3, 6].

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Moreover, chronic infection and inflammation increase the risk of developing MM and thus might be involved in its pathogenesis and progression [7, 8]. However, until now, the fundamental molecular mechanisms of these processes have not been clearly decoded [2, 4, 9].

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Toll-like receptors (TLRs) are non-catalytic, transmembrane, single receptors that link at a molecular level infection, tissue injury and inflammation [10, 11]. They stimulate the organism to respond with both innate and adaptive immune responses and increase tumor resistance and invasiveness. MM cells express TLRs and this shows that the bone marrow environment responds to tumor-induced signals with inflammation [2, 3]. However, most studies that have so far found a connection between TLRs and cancer have been restricted to mRNA studies: the mRNA level differences explain the variability of TLRs

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expressed in various cells and cellular responses. [12] Also, the expression patterns of the functional forms of these proteins are insufficiently known. Since TLRs modulate adaptive immune responses, current research is focused on TLR based therapeutic approaches that enhance the efficiency of anticancer immunotherapies [13]. TLRs and their distinct signaling pathways in myeloma cells might be potential

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therapeutic targets for tumor progression inhibitors [8]. In this review, we summarize the main advances in the mechanisms of TLR involvement in homeostasis. The paper supports the link between TLRs activation and cell proliferation and indicates that TLR overexpression might be involved in MM

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development. We focused on the role of TLRs in inflammatory processes present in MM and tried to emphasize their relation to tumor progression. Summary of Toll-like Receptors TLRs are members of the Pattern Recognition Receptor family (PRR), a group of proteins that facilitate the accurate identification of preserved Pathogen-Associated Molecular Patterns (PAMPs) [14-17]. They are type-I transmembrane glycoproteins expressed preferentially in the cells of the innate immune system, but also in platelets and B and T lymphocytes [18]. TLRs are expressed in myeloma cells and nonhematopoietic cells, where they may influence tumor growth and host immune responses [19].

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At present, 13 TLRs have been discovered, out of which the first 10 in humans and the remaining ones in mice [20]. All of them consist of three major domains: extracellular, transmembrane and cytoplasmic. The extracellular domain harbors a leucine rich repeat consisting of 19–25 tandem copies of the “xLxxLxLxx” motif [21] that detects and binds bacterial cell wall components, viral single or double

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stranded RNAs and small molecules of anti-viral compounds. The transmembrane domain is embedded in the cell membrane or the endosomal membrane. Lastly, the cytoplasmic domain, or Toll-IL1 receptor domain (TIR domain), binds to the adaptors MyD88, TIRAP/Mal, TRIF and TRAM and initiates the downstream signaling pathway [22-26].

TLRs play vital roles in the structural recognition of the innate immunity [27, 28]: bacterial membrane

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associated PAMPs activate TLR1, 2, 4, 5 and 6 on the plasma membrane [5], while nucleic acid based PAMPs from bacteria and viruses activate TLR3, 4, 7 and 9 expressed only on endosomes [28] (Table 1).

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TLR activation triggers the adaptive immune response. Here we present the roles of individual TLRs: TLR1 and TLR6 are active only after they form heterodimers with TLR2. The TLR1/2 heterodimer is a receptor for lipopeptides from mycobacteria and meningococci and the TLR2/6 heterodimer is a receptor for Mycoplasma lipoproteins and peptidoglycans [27, 29]. Overexpression of TLR1 and TLR2 on MM cells enhances their adhesion to bone marrow stromal cells and their sensitivity to Bortezomib and neutralizes the protective effect of bone marrow stromal cells over MM cells [30]. TLR2 forms heterodimers with either TLR1 or TLR6. These heterodimers respond to triacylated or diacylated bacterial

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lipopeptides. The TLR3 homodimers directly or indirectly bond with their ligands through ionic and hydrogen bonds [21]. They are activated by double-stranded RNA derived from viruses and stimulate the cell to produce IFNα/β and IL12p70 [31]. Fibroblasts express TLR3 on the cell surface, but myeloidderived dendritic cells express it intracellularly [31]. TLR3 and TLR9 could induce apoptosis and

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enhance immune responses against MM cells [32]. TLR4 is triggered by lipopolysaccharides from the plasma membrane of Gram-negative bacteria. It is a rapid pathogen sensor for in vitro studies and it stimulates rapid proliferation and survival of CD4+ T-cells [33]. Overexpression of TLR4 has numerous

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pro-tumor effects, including: increased cell proliferation, survival and evasion from immune surveillance [3]. TLR5 recognizes flagellin [34], modulates the transcription of TNFα in rheumatoid arthritis [35] and if overexpressed, it promotes chemoresistence [36]. TLR7 induces neurodegeneration if activated by microRNA let-7 [37] and together with TLR8 activates NF-κB after binding viral RNA. [38] TLR8 also mediates nervous tissue inflammation after an ischemic stroke attack [39]. Cleaved TLR9 combined with the N-terminal half of the ectodomain senses self DNA and prevents autoimmune diseases [40]. Deletion of the gene encoding for this receptor promotes atherosclerosis in ApoE deficient mice [41]. TLR10 is a co-receptor for TLR2 and mediates cellular responses to gram positive bacterial peptidoglycan [42] and

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influenza virus [43]. TLR11-TLR13 are expressed and functional in mice but not in humans. Even though TLR11 also exists in humans, it is only a pseudogene [44]. Upon binding to TLRs, the ligands trigger two major pathways: in the MyD88 dependent one, they activate the Nuclear Factor-κB (NF-κB), the Mitogen-Activated Protein Kinases (MAPKs) p38 and c-Jun

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N-terminal Kinase (JNK); in the MyD88 independent one, they activate both NF-κB and Interferon Regulatory Factor-3 (IRF3) [16, 45].

Toll-Like Receptors in inflammation

Immune dysfunction is a hallmark of myeloma. Intrinsic or therapy-related immunosuppression increases

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risk of recurrent infections [95] the main cause of mortality for the patients, [46] including MM patients [47-49]. MM is associated with infections caused by Gram-positive bacteria, of which Streptococcus

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pneumoniae predominates during the initial phase after diagnosis [50]. Inflammation represents an adaptive response to infection, trauma and homeostasis dysfunctions and can induce tumourigenesis due to chronic tissue damage and repair [4, 28]. Several types of Pattern Recognition Receptors on the cells of the immune system and myeloma cells can trigger the immune response to infection and malignancy. TLRs are the best characterized members of this protein family [51] and they influence tumor growth and host immune responses [2]. TLR expression patterns in MM cells and cells from the tumor microenvironment have numerous characteristics: MM cell lines express high levels of TLR1, TLR7,

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TLR8 and TLR9, moderate levels of TLR4, only mRNA for TLR2 and TLR5 and high levels of TLR3 proteins in spite of low levels of TLR3 mRNA [2]; human B lymphocytes strongly express TLR7, TLR9 and TLR10 and do not express TLR3, TLR4 and TLR8 [52]; human mesenchymal stromal cells express mRNA and proteins levels comparable to hematopoietic cells and macrophages only for TLR3 and TLR4

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and low levels of mRNA for TLR5, TLR6 and TLR9 [53]. Manier et al [54] reviewed the specific interactions between malignant plasma cells and the bone marrow microenvironment, while Bohnhorst et al [55] documented the TLR-mediated interactions between them.

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TLRs stimulate cell proliferation and link inflammation and cancer progression in MM [5]. The causative mechanisms of this phenomenon are not well understood, although some key events in the occurrence and progression of a tumor take place at the site of inflammation [56]. Inflammatory cells and molecules of the tumor microenvironment stimulate cell development [28]; inhibitory cytokines, inflammatory mediators, proteinases and nitric oxide enable cells to escape the immune system; inflammatory cells sustain tumor cell proliferation, survival, and migration and shape the its microenvironment [32]. Inflammation can have either tumor-promoting or tumor-suppressive effects depending on the type of inflammation (Table 2). Myeloma cells and plasma cells produce and secrete IL15 into their environment, a process through which they promote proliferation and cell survival of both immune and cancer cells

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[59]. The cells near the inflammation site activate the transcription factor NF-κB, which regulates both proinflammatory cytokine (secreted by T-helper cells) expression and T-helper 17 cell differentiation [57]. Proinflammatory cytokines contribute to the clonal expansion of neoplastic plasmocytes [58]. In MM patients, the stromal cells of bone marrow produce large quantities of IL6 and IL12, which promote

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T-helper cell growth and differentiation [58]. TLRs increase the tumor cell’s resistance to apoptosis and invasiveness. Thus, tumor immunology plays a key part of the ongoing effort to improve myeloma treatments.

TLR signaling

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The intracellular signals and cellular outcomes differ among TLRs (Fig 1.). Once TLRs bind to their ligands, they trigger two major signaling pathways [60]: the MyD88 dependent production and activation

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of NF-κB or the MyD88 independent production of type I interferon [16, 61]. However, both pathways are similar downstream of NF-κB. In the MyD88 dependent signaling pathway, MyD88 recruits Interleukin-1 receptor associated kinase 4 (IRAK4), which is internalized and phosphorylates IRAK1. Both IRAK1 and IRAK4 detach from MyD88 and bind to E3 ubiquitin ligase, TNF receptor associated Factor 6 (TRAF6), ubiquitin and the NF-kappa-B essential modulator [62] (Fig 2.). The MyD88 dependent pathway activates NF-κB and MAP kinases in response to all TLR ligands except TLR3 [63]. In the TIR-Domain-Containing Adapter-Inducing Interferon-Β (TRIF) dependent pathway (or MyD88

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independent pathway), TRIF binds to either TRAF6 or receptor interacting protein-1 and activates the TGF-beta activated kinase 1 end of NF-kB. NF-kB phosphorylates JNK and p38, which are further transported into the nucleus. Here, JNK and p38 phosphorylate and activate c-Jun and c-Fos. c-Jun and cFos form homodimers or heterodimers through their leucine-Zipper domains, which further form the AP1

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transcription factor. AP1 mediates the TNFα, IL6, and FAS transcription, regulates macrophage differentiation and upregulates TNFα, IL1, IL6 or IL12 production [64]. Autocrine secretion of interleukins stimulates TLR7 and initiates the STAT3 pathway, through which it activates B cells [60].

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Additionally, the TRIF dependent pathway triggers the IkappaB kinase complex (IKK 1/TBK1), which activates the IRF3 and IRF7 transcription factors that produce type 1 interferon [65]. LPS and CpG promote cell proliferation and survival through the MyD88 and MAPK signaling pathways. TLR signaling activates NF-κB and modulates the innate and adaptive immunity, the survival and proliferation of various cells, the cellular responses to stress, cancer progression and inflammation [1]. NF-κB can enter the nucleus and promote the IL6, IL18, TNFα and IFNα gene transcription. An increase in the level of IL6 and IL18 are clinically relevant in multiple myeloma. IL6 modulates the growth and survival of MM cells. Two distinct pathways control these processes: the MAPK cascade controls cell growth P38 MAPKs control apoptosis and IFNα mediates it and the STAT3 pathway prevents apoptosis

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[66]. Normal human B cells express lymphocyte maturation protein 1 (BLIMP1) in reaction to antigens [67] and differentiate into plasma cells. The prdm1 gene sequence encoding for BLIMP1 contains binding sites for NF-κB. This protein increases the lifespan of myeloma cells in the bone marrow [68]. Studies on bone marrow extracted from MM patients have proven that they have increased expression of TLR1,

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TLR4, and TLR9. At diagnosis, macrophages and monocytes express predominantly TLR2 and TLR6 and produce most of the inflammatory cytokines in the myeloma environment [69]. TLR activation induces IL6 synthesis [3, 5]. TLR ligands stimulate cell growth and survival in MM patients, partially due to an autocrine production of IL6. Thus, TLRs may help maintain and progress MM [55].

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TLR based therapies in MM

MM is still an incurable disease and there is a need for novel ways that induce long-term tumor regression

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and improve the disease outcome [63]. Current conventional MM therapies imply the use of cytotoxic agents (especially the alkylating agent melphalan) combined with corticoids and more recently with the proteasome inhibitor Bortezomib. A frequently used effective treatment for MM in relatively young and fit patients is high-dose chemotherapy followed by autologous stem cell transplant [70]. This procedure efficiently reduces the plasma cell number, but the disease relapses in virtually 100% of cases because malignant stem cells are resistant to chemotherapy. Hawley S et al [71] proved that the tumor propagating cells in multiple myeloma have stem cell like properties that confer their resistance to chemotherapeutic

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agents [71]. Recently, Staudinger et al [72] generated a novel immunotoxin by genetic fusion of a CD317 specific single chain Fv (SvFv) antibody and a truncated variant of Pseudomonas aeruginosa endotoxin A (ETA). HM 1.24-ETA inhibited the growth of both IL6 dependent and independent myeloma cell lines and it was not cytotoxic against CD137 positive cells from healthy tissue. This shows that CD317 may

tumors [72].

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represent a promising target for specific and efficient immunotoxin therapy in patients with plasma cell

As traditional therapies have little or no documented participation in TLR signaling pathways, with the

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notable exception of corticosteroids [73], a possible way to overcome resistance to chemotherapy [74] is to develop immunotherapy approaches that target and remove early, quiescent myeloma cells [75]. However, TLR-based therapies sensitizes myeloma to traditional treatments .Therefore, new therapies for relapsed MM are required [77]. TLRs 2, 4, 5 and 9 are expressed on the surface of many tumor cells [78]. Their ligands interact directly with cancer cells, promote tumor progression and make cells less vulnerable to chemotherapy. Agonists and antagonists of the TLR signaling pathway (TIR-8 activators, IKK-2 inhibitors, non-steroidal anti-inflammatory drugs, glucocorticoids or parthenolide) can directly or indirectly inhibit tumor development [79]. Since TLR signaling manipulation might better control infection and regulate inflammation, TLRs could be a target for host modulation in multiple myeloma

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[56]. At present, many TLR agonists are under development in animal studies or clinical trials in order to establish their anti-tumor action. J. Abadi et al [7] showed that the TLR1 ligand Pam3CSK4 stimulates TLR1/2 and promotes human myeloma cell lines adhesion to fibronectin. Combined with Velcade (bortezomib), it enhances the caspase 3 activity in these cells and induces the death of fibronectin-

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adherent MM cells [7]. TLR3 agonists initiate the NF-kB pathway and produce type I interferons that trigger antiviral barriers that efficiently destroy cancerous cells [80]. The modified TLR3 ligand CpGKSK 13 inhibits osteoclast formation in both mouse and human cell cultures. This effect downregulates the triggering receptor expressed on myeloid cells 2 expression in osteoclast precursors and provides the possibility to use TLR3 modified ligands as anti-osteoclastogenic therapeutic agents [80].

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Monophosphoryl lipid A is a partial agonist of the TRAM/TRIF pathway that stimulates TLR4 and inhibits Mal/MyD88 signaling [81]. The flagellin derivative CBLB502 is a TLR5 agonist that activates

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NF-κB and has reduced immunogenic characteristics [82]. Myeloma cell lines treated with flagellin activate NF-κB through P38 and PI3k/AkT signaling and show increased proliferation, IgG production and IL6 expression. Flagellin treatment inhibits caspase and PARP activity and renders MM cells resistance to apoptosis and doxorubicin [36]. Imidazoquinolines are antiviral agents and TLR7/8 agonists that stimulate cytokine production. Another TLR7/8 agonist, IPH-3201, could be used to treat cancer, infectious and autoimmune diseases [83]. A phase 1 clinical trial on the TLR 7 agonist 852A (S-32865) in patients with hematological malignancies showed that the drug was well tolerated and associated with

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measurable immune activation (increased NK and T-cell activation)[84]. New human cell specific CpG(A)-STAT3 siRNA conjugates can induce TLR9-dependent gene silencing and activate primary immune cells (myeloid dendritic cells, plasmocytoid dendritic cells and B cells) in vitro. Specific CpG(A)-siRNAs effectively knock down the expression of the oncogenic proteins STAT3 and BCL-

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X(L) in TLR9 positive hematologic tumor cells in vivo. These results emphasize the potential of using immunostimulatory CpG-siRNA oligonucleotide as a novel therapeutic strategy for hematological malignancies [85]. Another TLR9 agonist, C792, inhibits myeloma cell growth induced by plasmacytoid

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dendritic cells, promotes apoptosis and enhances bortezomib cytotoxicity. C792 restores the ability of MM patient-derived plasmocytoid dendritic cells to stimulate T-cell proliferation. It enhances the antitumoral activity of bortezomib, lenalidomide, SAHA (vorinostat) and melphalan [76]. Intracellular signaling kinases are further recommended as drug targets for negative regulation of TLR signaling. Serine or threonine glycogen synthase kinase-3 efficiently downregulates the TLR-mediated responses and inflammatory responses [86]. Mechanically inhibiting glycogen synthase kinase-3 induces the cAMP response element-binding protein to bind to the p65 subunit of NF-κB. It upregulates the cAMP response element-binding protein dependent production of interleukins, as well as the NF-κB dependent inhibition of proinflammatory cytokines [56]. Inhibiting ligand binding could be a probable

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target for the TIR domain pathway. This would inactivate specific TLRs and block signal transduction [28, 56, 79]. Clinical trials on TLR agonists have increased in number in recent years (source: www.clinicaltrials.gov). Table 3 displays a list of clinical trials on TLR agonists in cancer treatment, most of which are in phase I or II of study. Most of the selected trials are still ongoing and several drugs have

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already shown positive results or have been approved as treatment of cancers. However, most of the listed drugs are used as adjuvants yet and none of them in MM. Also, none of the TLR-based drugs were first developed against this pathology. The large number of ongoing clinical trials on TLR agonists in cancers indicates the possible successful use of these drugs in treating MM.

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Future prospects

Strong expression of TLRs in human myeloma cell lines and primary tumor cells suggests a response to

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tumor-induced inflammatory signals in the bone marrow environment [2]. TLRs are expressed heterogeneously on myeloma cells and cell lines and their expression is significantly higher than in normal plasma cells. A study of several human myeloma cell lines and primary tumor cells showed no correlation between TLR expression at mRNA and at protein levels: all cell lines and primary cells expressed both mRNA and proteins for TLR1, TLR3, TLR4, TLR7, TLR8, and TLR9; human myeloma cell lines expressed only mRNA and primary tumor cells expressed only low protein levels of TLR2 and TLR5 [2]. As the signaling pathways via TLRs are now being unveiled, there are still several unanswered

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questions about the mechanism that regulates the TLR-mediated gene induction. For example, is it possible that there might be an additional pathway downstream of MyD88 that continues their signaling cascade? TLR-induced NF-κB has been demonstrated to regulate a subset of TLR-inducible genes [87]. In this model, TLR stimulation immediately induce NF-κB, which activates the production of IL6,

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IL12p40 and granulocyte-macrophage colony-stimulating factor in macrophages. It would be interesting to examine whether this two-step TLR-mediated gene induction model can be applied or not to other subsets of genes induced by TLRs. An extensive knowledge of the mechanisms of the innate immunity is

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helpful in order to develop innovative therapies to manipulate immune responses in both malignant and infectious diseases [88]. These details would provide a link to understand diverse pathways in MM pathogenesis, progression and preservation [55]. Furthermore, the combination of imaging, systems biology and immunology may reveal the dynamics of TLR-mediated responses and their role in multiple myeloma and different autoimmune diseases. TLR expression profiling could be a strategy to identify certain cancers [89-91], as TLR-mediated signaling promotes tumor growth [78]. Moreover, various TLR agonists or antagonists are investigated in different types of cancers [92]. Given that specific TLRs or their pathways mediate both cancer pathogenesis and anti-cancer immune response, it is still unclear whether it is the agonists or antagonists

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of TLRs that are the promising therapeutic approaches. In the future, it will be important to test if downregulation of TLRs on MM cells could be a viable targeting approach, such as the antiproliferative effects of TRAF6 signaling (downstream to TLR pathway) on myeloma cells [96]. Better describing the interaction profiles of the existing chemotherapeutics at the level of TLRs and TLR-dependent pathways

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should help us optimize therapeutic schemes. Also, it is important to characterize the expression profiles of TLRs in different populations of MM patients and evaluate how do different expression profiles influence the patient’s chemosensitivity. The goal of personalized medicine based on TLR profiles may accelerate the search for new potential immune-based chemotherapeutics.

This review emphasizes the effects of TLRs stimulation by endogenous ligands on tumor cells. Also, the

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field of TLR-based adjuvants for immunotherapy remains in development: several agonists of TLR2-9 were tested as adjuvants for vaccine therapy in infectious diseases and cancer. Of them, the agonists of

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TLR 4, 7/8 and 9 showed increased efficacy in clinical trials and were approved as adjuvants for vaccine therapy [94]. The research on TLRs remains at an early stage, so we need detailed information on the role of each TLR in various types of cancers in order to develop novel TLRs based therapeutic strategies [16]. Furthermore, in the period of very expensive drugs, the innovative treatment approach with inexpensive TLR based myeloma therapy is always welcome.

Conclusion

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MM is a life-threatening B cell malignancy for which regular therapy is inefficient. The extremely complex pathogenesis of MM gives us a chance to encourage novel drugs and approaches with various mechanisms of action based on targeted therapy. TLRs act as radars of the innate immunity: they detect various pathogens and activate both the adaptive immunity and normal B lymphocytes. Each TLR

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triggers diverse signaling pathways and has discrete biological effects. TLR stimulation upregulates IL6 through the NF-kB pathway and induces MM cell growth and survival. The processes started by TLR activation go beyond the response of the innate immunity: through controlling the cellular outcomes to

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stress and inflammation, TLR influence cancer cell proliferation and tumor progression. TLRs are future therapy targets and interference with their signaling pathway limits tumor formation. Enhancing their activity could provide an adjuvant therapy to standard treatments. The combination of imaging techniques, systems biology and immunology will expose the particular aspects of TLR-mediated responses and their role in multiple myeloma as well as in non-malignant autoimmune conditions.

Conflict of interest The authors have declared no conflict of interest.

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Acknowledgements

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The authors would like to acknowledge Dr. Mangala Lahkar, Coordinator of IBT-Hub NIPER Guwahati for providing support in every manner. The authors would also like to thank NIPER Guwahati staff for their helpful discussion.

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ACCEPTED MANUSCRIPT Table 1. Overview of TLRs with their respective function

TLRs

Species

Location

PAMPs recognized by TLRs

Function

TLR1/2

H/M

Cell surface

Ac3LP, Glycolipids

receptors for lipopeptides

Triacyllipopeptides H/M

Cell surface

Ac2LP,

LTA,

Zymosan, receptors for lipopeptides

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TLR2/6

Peptidoglycan, Diacyllipopeptides TLR3

H/M

Endosome

polyI:C, dsRNA(reovirus), RSV, stimulates MCMV

H/M

TLR5

H/M

Cell surface

Cell surface

and IL-

12p70 production

LPS, Taxol, Heparan, Hyaluronate,

stimulates rapid proliferation

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TLR4

IFN-α/β

F-prost, VSV, Envprost,

and survival of CD4+ T-cells.

Flagellin

modulates the transcription of

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TNFα in rheumatoid arthritis

H/M

Endosome

ssRNA, imiquimod, loxoribine, and activates NF-κB, increases guanine analogs such as antibody secretion and cytokine loxoribine production in B cells

TLR8

H/M

Endosome

ssRNA, IAQ (R848)

H/M

Endosome

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TLR9

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TLR7

TLR10

TLR11

H

M

Cell surface

Cell surface

activates NF-κB, mediates the inflammation of the nervous tissue after an ischemic stroke attack

dsDNA viruses (HSV, MCMV), increases proliferation, survival CpG

and differentiation, as well as

motifs from bacteria and viruses,

secretion

hemozoin (plasmodium)

immunoglobulins, autoimmune

of

IL-6,

IL-10,

disease prevention Unknown

modulates cellular responses to gram

positive

bacterial

peptidoglycan Profiling

expressed

and

completely

functional in mice TLR12

M

Cell surface

Unknown

expressed

and

completely

functional in mice TLR13

M

Endosome (Probably)

Unknown

expressed

and

functional in mice

completely

ACCEPTED MANUSCRIPT H- Human, M-Mouse, PAMP- pathogen-associated molecular patterns; LTA- lipoteichoic acid; RSV- respiratory syncytial virus; MCMV- murine cytomegalovirus; LPS- lipopolysaccharide; CpGcytosine-guanine dinucleotide; R848- Resiquimod, VSV- vesicular stomatitis virus; ssRNA- single-

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stranded RNA

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Table 2. The dual activity of TLRs

3,4 4 4,5

TLR

Suppression of angiogenesis Development of Apoptosis Increase in chemosesitivity Inhibition of T antigen presentation

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Activation of regulatory T-cells

2,9

Antitumor activity

7,9

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TLR

3,4,7,9 2,4,7

4,5,7,8,9

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Tumor stimulating activity Stimulation of Angiogenesis Stimulation of Proliferation Chemoresistance

ACCEPTED MANUSCRIPT Table 3. Relevant TLR-targeted drugs in clinical trials or approved for treatment Compound

Target

Indication

Status

Imiquimod

TLR7 [97]

approved by FDA

bacillus Calmette–Guérin (BCG) GNKG168 (CpG 685)

TLR2, TLR4 [97]

basal and squamous cell carcinoma bladder cancer

NCT00276159

852A

TLR7 [99]

NCT01308762

IMM-101

TLR2 [101]

NCT02100618 NCT00541970

AS04

TLR4 [102]

NCT01853878

AS15

NCT01079741

IMO-8400

NCT01079741 NCT01920191 NCT01532960 NCT01834248

Hiltonol (PolyICLC)

TLR4 and TLR9 [103] TLR7, TLR8 and TLR9 [104] TLR3 [97]

NCT0072905

IMO-2055 (IMOxine®)

NCT0003127

CpG 7909 (PF-3512676)

TLR9 [105]

TLR9 [106]

Notes

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approved by FDA Phase I

ongoing

Phase II

limited number of patients included

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acute lymphoblastic leukemia, acute myelogenous leukemia hematological malignancies, gynecological cancers, melanoma melanoma, B-cell lymphoma adjuvant for HPV16/18 L1 VLP AS04 vaccine non-small cell lung cancer Waldenstrom's Macroglobulinemia

Phase III

safe and well tolerated ongoing

Phase I/II

ongoing

Phase II

ongoing

adjuvant for various cancers (melanoma, glioma, ovarian cancer, hematological malignancies) renal cell carcinoma

Phase I/II

ongoing

Phase II

breast cancer

Phase II

completed, no results published completed, no results published

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TLR9 [98]

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NCT01743807

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Clinical trial identifier

Phase I

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Fig. 1: The intracellular signals and cellular outcomes of TLRs.

The red straight arrow shows the MyD88 dependent, MAPK dependent pathway; The red dashed arrow shows the MyD88 dependent, MAPK independent pathway; The green straight arrow shows the MyD88 independent, MAPK dependent pathway; The green dashed arrow shows the MyD88 independent,

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MAPK independent pathway.

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Fig. 2: The location of different TLRs along with their signalling cascade in cancerous B cells. TLR- Toll like receptor; IRAK- Interlukin-1 receptor associated kinase; MyD88- Myeloid

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differentiation primary response 88; TRAF- TNF receptor associated Factor; IKK- IkappaB kinase; NF-κB- Nuclear Factor kB; AP1- Activator protein 1; TRIF- TIR-domain-containing

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adapter-inducing interferon-β.

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TLRs are key components in inflammatory processes

•

TLR activation stimulates cell proliferation in chronic inflammation

•

We hypothesize that TLR activation might trigger and maintain MM

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We believe that ongoing and future TLR based therapies could treat MM

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•

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Highlights: