Accepted Manuscript

The role of G protein-coupled receptors in lymphoid malignancies

Adrienne Nugent, Richard L. Proia

PII: S0898-6568(17)30218-8 DOI: doi: 10.1016/j.cellsig.2017.08.002 Reference: CLS 8973 To appear in: Cellular Signalling Received date: 25 May 2017 Revised date: 4 August 2017 Accepted date: 7 August 2017

Please cite this article as: Adrienne Nugent, Richard L. Proia , The role of G protein- coupled receptors in lymphoid malignancies, Cellular Signalling (2017), doi: 10.1016/ j.cellsig.2017.08.002

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Title: The role of G protein-coupled receptors in lymphoid malignancies

Adrienne Nugent and Richard L. Proia

National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD, 20892, USA; [email protected] and [email protected]

Address correspondence to: Richard L. Proia, PhD NIH/NIDDK 10 Center Drive, Bethesda, MD, 20892, USA (301) 496-4391 [email protected]

Disclosure of conflicts of interest: None

Author contributions: AN conceptualized, wrote and edited the review; RLP conceptualized and edited the review. All authors have approved the final article.

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ABSTRACT:

B cell lymphoma consists of multiple individual diseases arising throughout the lifespan of B cell development. From pro-B cells in the bone marrow, through circulating mature memory B cells, each stage of B cell development is prone to oncogenic mutation and transformation, which can lead to a corresponding lymphoma. Therapies designed against individual types of lymphoma often target features that differ between malignant cells and the corresponding normal cells from which they arise. These genetic changes between tumor and normal cells can include oncogene activation, tumor suppressor gene repression and modified cell surface receptor expression. G protein-coupled receptors (GPCRs) are an important class of cell surface receptors that represent an ideal target for lymphoma therapeutics. GPCRs bind a wide range of ligands to relay extracellular signals through G protein-mediated signaling cascades. Each lymphoma subgroup expresses a unique pattern of GPCRs and efforts are underway to fully characterize these patterns at the genetic level. Aberrations such as overexpression, deletion and mutation of GPCRs have been characterized as having causative roles in lymphoma and such studies describing GPCRs in B cell lymphomas are summarized here.

Keywords: GPCR; lymphoma; cancer; mutation; chemokine receptors; lipid receptors

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Abbreviations: ABC = Activated B cell-like ALL = Acute lymphoblastic leukemia ATP = Adenosine triphosphate B-ALL = B cell acute lymphoblastic leukemia BCR = B cell receptor BL = Burkitt lymphoma BLR1 = Burkitt lymphoma receptor 1 cAMP = Cyclic adenosine monophosphate CCND1 = Cyclin D1 CLL = Chronic lymphocytic leukemia CREB = Cyclic adenosine monophosphate response element-binding protein CRLF2 = Cytokine receptor like factor 2 DLBCL = Diffuse large B cell lymphoma DLBCL-NOS = Diffuse large B cell lymphoma-not otherwise specified EBI2 = Epstein-Barr virus-induced G protein-coupled receptor 2 FL = Follicular lymphoma GCB = Germinal center B cell-like GPCR = G protein-coupled receptor HCL = Hairy cell leukemia IGHV = Immunoglobulin heavy-chain variable region gene IgM = Immunoglobulin M IHC = Immunohistochemistry JNK = c-Jun N-terminal kinase LPA = Lysophosphatidic acid LPL = Lymphoplasmacytic lymphoma MALT = Mucosa-associated lymphoid tissue MAML2 = Mastermind like transcriptional coactivator 2 MAPK = Mitogen-activated protein kinase MCL = Mantle cell lymphoma MFI = Mean or median fluorescence intensity MGUS = Monoclonal gammopathy of undetermined significance MMP-9 = Matrix metallopeptidase 9 MZL = Marginal zone lymphoma NF-κB = Nuclear factor-kappa B NHL = Non-Hodgkin’s lymphoma PMBCL = Primary mediastinal large B-cell lymphoma S1P = SphingosineACCEPTED-1-phosphate MANUSCRIPT SLL = Small lymphocytic lymphoma T-ALL = T cell acute lymphoblastic leukemia TFH = Follicular helper T cell Treg = Regulatory T cell VEGF = Vascular endothelial growth factor VLA-4 = Very late antigen-4 WM = Waldenstrom macroglobulinemia

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1. Introduction

B cell lymphomas encompass a variety of neoplasms affecting lymphocytes in the immune system. In all cases, a normal B cell acquires a set of mutations or genetic aberrations that result in clonal proliferation of malignant cells. The stage of development a B cell is in when it acquires these genetic alterations dictates the subsequent lymphoma that develops. Through advances such as next generation sequencing and high throughput screening technologies, the underlying genetics and mechanisms of lymphomagenesis are becoming more clearly defined.

GPCRs are seven-transmembrane domain cell surface-spanning receptors that regulate and transmit extracellular signals to induce numerous intracellular signaling pathways. There are hundreds of known GPCRs, each of which binds a specific ligand or set of ligands, which then induce conformational changes that lead to downstream signaling events. The cell surface ligand-binding capabilities of GPCRs make them highly desirable as drug targets for oncogenic cells. A complete and thorough understanding of GPCR expression in normal and malignant cells, coupled with a detailed knowledge of the molecules that bind to each GPCR and the downstream signaling pathways they activate, will reveal numerous opportunities for targeted therapeutics to improve disease outcome. A summary of what is known regarding GPCR expression and mutation in B cell lymphomas is reviewed here and outlined in Figure 1.

2. B-cell Acute Lymphoblastic Leukemia Acute lymphoblastic leukemia (ALL) is a neoplasm of the lymphoid precursor cells and is characterized as either B cell ALL (B-ALL) or T cell ALL (T-ALL) based on the cell of origin. B-ALL is the most common childhood malignancy and occurs less frequently in adults [1]. B-ALL cases are often subgrouped based on cell of origin, patient age, or the presence of a chromosomal aberration.

Multiple chemokine receptors are expressed in B-ALL and these findings are summarized in Table 1. Chemokine receptor expression frequently varies between lineage-derived subgroups of B-ALL and this can provide insight into the underlying function of GPCRs in B-cell development and lymphomagenesis. For example, CXCR1 surface protein was not found in pro-B or pre-B ALL but was present in 5/17 (29%) cases of early pre-B and 3/5 (60%) cases of B-ALL [2]. Meanwhile, flow cytometry of CXCR2 and CXCR3 found these receptors to be expressed in all early pre-B, pre-B and B-ALL samples tested along with more than half of pro-B samples [2, 3]. However, another study that usedACCEPTED immunohistochemistry (IHC MANUSCRIPT) instead of flow cytometry to detect surface protein only found CXCR3 in 3/9 (33%) lymphoblastic leukemia/lymphoma patients [4].

CXCR4 is the most frequently studied chemokine receptor in lymphoma and its role in signaling in B-ALL has been extensively reviewed [5]. CXCR4 is normally expressed on pre-B and mature B cells and is essential for migration in lymphoma [4]. Elevated mRNA or protein expression of CXCR4 in B-ALL has been correlated with unfavorable clinical outcome [6], extramedullary organ invasiveness [7, 8], and time point and site of relapse [9]. CXCR4 has been identified in the majority of pro-B, pre-B and B-ALL cases and in

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some early pre-B cases [2, 3, 8, 10]. The estrogen receptor GPER1 (also known as GPR30) is also believed to regulate CXCR4 signaling in pre-B cell ALL [11].

CXCR5 is known to play a role in signaling and cell migration in lymphoma, however there are conflicting reports regarding its cell surface expression in B-ALL. Two flow cytometry studies did not identify any precursor-B, pro-B or pre-B patients with CXCR5 expressed [3, 10] while a third study found CXCR5+ cases in 3/9 (33%) pro-B, 5/17 (29%) early pre-B, 4/12 (33%) pre-B and 5/5 (100%) B-ALL cases [2]. A fourth report identified CXCR5 expression only on CD23+CD5+ B-ALL cells [12], while a final study found the receptor to be expressed in all B-ALL cases [4].

Other chemokine receptors that have been detected by flow cytometry in B-ALL include CCR3 and CCR4 [2, 12] as well as CXCR7 (also known as ACKR3), which is strongly upregulated in the bone marrow in B-ALL compared to normal tissue and plays a role in controlling CXCR4-mediated migration [13]. CCR7 cell surface expression has been found to vary between studies [2, 3, 12] while CCR1, CCR2, CCR5 and CCR6 were extremely rarely or never expressed [2, 3, 10, 14].

In addition to grouping cases of B-ALL by cell of origin, these lymphomas can also be subcharacterized by genetic rearrangements. Fusion of the purinergic receptor P2RY8 promoter to the CRLF2 (cytokine receptor like factor 2) gene frequently occurs in B-ALL but reports of the functional consequences of this rearrangement have been conflicting. Although P2RY8-CRLF2 fusions were significantly correlated with poor outcome in adolescent and adult patients [15, 16], the consequences of the fusion in pediatric B-ALL is less clear as some studies have found it to be associated with poor outcome [17-19] whereas others have not found any significant correlation [20, 21]. A meta-analysis of these studies concluded that presence of the P2RY8-CRLF2 fusion was in fact significantly associated with poor prognosis of relapse-free survival and event-free survival [22]. Another finding among these studies was that P2RY8-CRLF2 was more common in B-ALL patients with trisomy 21 [19, 23-26] or intrachromosomal amplification of chromosome 21 [27] than those with normal chromosome 21 [1, 18-21, 25, 26, 28-30].

P2RY8-CRLF2 rearrangements were significantly enriched in patients harboring deletion of the lymphoid lineage-directing transcription factor IKZF1 in one study [31] but not correlated in another study, and this discrepancy is suggested to be correlated to ethnicity [1]. Interestingly, reduced protein expression of IKZF1 has also been associated with upregulation ofACCEPTED the orphan GPCR GPR132 MANUSCRIPT (also known as G2A) in B-ALL patients that do not harbor the BCR-ABL fusion, which encodes a tyrosine kinase oncoprotein [32]. In fact, mice transplanted with BCR-ABL transduced bone marrow deficient of GPR132 developed tumors more quickly and had accelerated tumor progression and death compared to those with wild type GPR132 suggesting that GPR132 acts as a negative regulator of BCR-ABL induced leukemogenesis [33].

Other GPCRs that have been described in B-ALL include oncogenic roles for the prostaglandin receptor PTGER2 [34-37] and orphan receptor GPR34 [38], increased

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protein expression of the thrombin receptor F2R (also known as PAR1) at significantly higher levels than lymphocytes from healthy donors [39] and decreased expression of S1PR1 and S1PR2 transcripts [40] and the tachykinin receptor TACR1 in tumor compared to normal B lymphocytes [41]. A summary of all GPCRs reported in B-ALL is shown in Table 2.

3. Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is a heterogeneous disease that arises from CD5+ B cells. CLL is the most frequently occurring leukemia in Western countries and has an extremely variable prognosis that can range from stable, indolent disease not requiring intervention to aggressive malignancy [42]. CLL is often characterized by immunoglobulin heavy-chain variable region gene (IGHV) mutation status. Unmutated IGHV CLL originates from pre-germinal center cells and is defined as having less than a 2% difference between leukemic clone and germline DNA sequence in the IGHV region. Mutated IGHV arises from post-germinal center cells and contains a 2% or greater difference between leukemic clone and germline IGHV sequence and has a significantly better prognosis than unmutated IGHV [43]. CLL that localizes to lymph nodes is also known as small lymphocytic lymphoma (SLL) and represents a subset of CLL.

A variety of receptors spanning multiple GPCR families have been studied in CLL and recent attempts to target members of the lipid receptors in vitro and in vivo have shown a range of success. The sphingosine-1-phosphate (S1P) receptors S1PR1 and S1PR2 transcripts were found to be downregulated in CLL compared to control B cells [40], with S1PR1 expression particularly reduced in unmutated IGHV CLL patients and S1PR2 impaired in both mutated and unmutated CLL [43]. This downregulation is thought to be due to cell interaction with the tumor microenvironment to regulate egress of malignant cells from the lymphoid tissues to peripheral blood [44]. Treatment with Syk, Btk, and B cell receptor (BCR) inhibitors has been effective at increasing S1PR1 protein expression to induce CLL cell mobilization into the blood so that cells are more sensitive to cytotoxic drugs [44-46].

Contrary to the downregulation of S1PR family GPCRs, CLL cells have increased mRNA expression of the lysophosphatidic acid (LPA) family receptors LPAR1, LPAR3 and LPAR4 compared to normal B cells [47]. Increased LPAR1 mRNA has been shown to be associated with more aggressive disease [47] and LPA signaling was found to act as a survival factor by protecting primary CLL cells from spontaneous and chemotherapy- induced apoptosis [48]. Further study revealed that treatment of B cell lines with LPA induced vascularACCEPTED endothelial growth factor MANUSCRIPT(VEGF) expression via activation of c-Jun N- terminal kinases (JNK) and nuclear factor-kappa B (NF-κB) and protected cells against apoptosis [47, 49].

Cannabinoid signaling pathways have been investigated for potentially containing novel therapeutic targets in CLL/SLL. The cannabinoid receptor transcripts CNR1 and CNR2 were found to be overexpressed in CLL and SLL compared to normal B cells and high CNR1 expression was significantly associated with shorter overall survival [50, 51]. Although treatment with cannabinoids reduced viability of CLL cells in culture, the

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simultaneous death of healthy cells suggested that targeting cannabinoid receptors could have poor therapeutic value [50].

Numerous GPCRs have significantly altered expression in CLL as compared to healthy lymphocytes and these expression patterns can serve as biomarkers of disease subtype or progression. For example, tachykinin receptor TACR1 mRNA is overexpressed in CLL patient cells compared to normal B lymphocytes and expression is higher in aggressive IGHV-unmutated CLL compared to indolent IGHV-mutated CLL [41]. Conversely, CLL mononuclear leukocytes contain fewer beta-2 adrenergic receptors (ADRB2) than healthy cells and increased dysfunction of the receptor complex is correlated with disease progression [52]. ADRB2 agonists have been shown to induce apoptotic cell death in CLL cells alone and synergistically with other agents [53] and expression of alpha-2 adrenergic receptors has also been described in CLL [54].

Multiple GPCRs are believed to affect cyclic adenosine monophosphate (cAMP) and calcium signaling in CLL. RNA transcripts from the adenosine receptors ADORA2A and ADORA2B and purinergic receptor P2RY11 were found to be expressed in CLL lymphocytes it is believed that adenosine induces cAMP accumulation via ADORA2A while adenosine triphosphate (ATP) induces cAMP through P2RY11 [55]. The calcitonin receptor CALCR mRNA and protein were shown to be overexpressed in CLL cells compared to healthy B cells and it is suspected that an increase in CALCR expression increases the concentration of intracellular calcium to promote lymphocyte activation and proliferation [56]. In addition, mRNA from the cysteinyl leukotriene receptor CYSLTR1 was found to be well-expressed in CD19+ CLL cells, albeit at lower levels than normal CD19+ cells, and was found to mediate intracellular calcium and cell migration in response to leukotrienes [57].

Notable oncogenic hallmarks such as increased DNA synthesis, cell cycle progression, and adaptation to the tumor microenvironment are all influenced by GPCRs in CLL. The endothelin receptor EDNRA was found to be overexpressed at both the mRNA and protein level in CLL cells compared to normal cells and activation of EDNRA via endothelin-1 resulted in increased proliferation, cell cycle progression and mitogen- activated protein kinase (MAPK) signaling [58]. The acid sensing GPCR GPR65 transcript levels in CLL were significantly correlated with expression of the apoptosis- regulating proteins Bcl-2, Mcl-1 and Bcl-xl, suggesting that GPR65 may aid CLL cells to survive in the acidic tumor microenvironment [59]. Finally, CLL cells express the leukotriene receptor LTB4R (BLT1) protein and treatment of these cells with leukotriene biosynthesis inhibitorsACCEPTED inhibited DNA synthesis MANUSCRIPT and antigen expression and thus represent a novel CLL therapeutic [60].

Other GPCRs notable for changes in expression on CLL cells include upregulation of the thromboxane A2 receptor TBXA2R mRNA [61] and up and downregulation of mRNA and protein from the neurotensin receptors NTSR2 and NTSR1, respectively [62].

The Eμ-TCL1 mouse model of CLL has been used to study multiple aspects of the disease including changes in gene expression during disease development. In this model,

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the gene expression of the Wnt/Fzd pathway GPCRs FZD6 and FZD1 both increased during leukemogenesis and knockout of FZD6 in the model resulted in a delay and decreased incidence of disease [63]. However, differences between mouse and human FZD6 have made it challenging to understand the relation of this finding to humans [64]. In a study of human mRNA, FZD3 was the only Fzd receptor that had significantly higher expression in mutated or unmutated CLL when compared to normal B cells [65]. FZD3 mRNA was also found to be overexpressed in a separate cohort of early-stage CLL patients compared to normal volunteers [66].

The expression levels of chemokine receptors in CLL and SLL are summarized in Table 1 and their role in disease has been reviewed elsewhere [67]. Of note, CXCR3 is highly expressed in CLL and plays a role in mediating cell chemotaxis. In one study, 31/31 (100%) CLL patients had CD5+ B cells positive for CXCR3 via flow cytometry compared to 0/6 (0%) healthy subjects [68] while in another study CXCR3 was positively identified via IHC in 34/36 (94%) CLL/SLL patients and by flow cytometry in 12/13 (92%) cases [4]. CXCR3 was also found to be overexpressed in low stage CLL compared to healthy B cells via gene expression profiling [66]. With regards to the consequences of CXCR3 expression, one study with a small cohort of 21 patients found no correlation between CXCR3 flow cytometry mean fluorescence intensity (MFI) and the number of lymphocytes or clinical stage of disease [69] whereas a larger study of 149 CLL patients found that patients with high CXCR3 and low CXCR4 MFI had a significantly lower risk of disease progression in both IGHV mutated and unmutated CLL [70].

CLL cells express an abundance of CXCR4 protein [3, 10, 46, 71-73] to enable migration and infiltration of bone marrow [74]. Increased protein levels of CXCR4 were found to be associated with survival in familial CLL [75] and lymphadenopathy [46] but it is unclear whether they do [76] or do not correlate with disease stage [77]. Multiple genetic aberrations of CXCR4 in CLL have been discovered including 3/186 (2%) familial cases of CLL containing a mutation in the coding region of the gene [78] and a case study of a translocation resulting in a CXCR4/MAML2 (mastermind like transcriptional coactivator 2) fusion protein [79].

As CLL cells do not express CXCR7 protein, all CXCL12-mediated responses are accomplished via CXCR4 and numerous downstream signaling molecules including activation of ZAP-70, MAPK and Akt signaling pathways [80-82]. CXCR4 was observed to be hyperphosphorylated in CLL compared to normal lymphocytes and inhibition of PIM kinase caused dephosphorylation and internalization of CXCR4 [83]. Numerous other therapeuticsACCEPTED targeting CXCR4 downregulation MANUSCRIPT have been investigated including suberoylanilide hydroxamic acid [84], AMD3100 (plerixafor) [85], the combination of CXCR4 antagonists and passive immunotherapy [86], PI3K inhibitors [87], small peptide inhibitors [88] and ibrutinib-mediated BTK inhibition [46, 89].

The chemokine receptor CXCR5 plays a significant role in CLL/SLL pathogenesis. The receptor protein has been identified on greater than 90% of patient samples [3, 4, 10, 72] and shown not to be affected by IGHV mutation status [90]. Depending on the study, CXCR5 protein was either similarly expressed or significantly overexpressed relative to

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normal CD5+ B cells [12, 71, 90] and more highly expressed in CLL compared to other B cell neoplasms [3, 10, 71, 90]. Downregulation of CXCR4 and CXCR5 via BCR signaling was found to correlate with unfavorable disease outcome [91, 92] and reduced CXCR5 MFI was observed in del 17p/11q CLL, which has a worse prognosis than normal CLL [93]. Experiments in cell lines revealed that overexpression of CXCR5 along with CCR7 in CLL and B-ALL enabled resistance to apoptosis via upregulation of PEG10 and stabilization of caspase-3 and caspase-8 [12, 94]. In concordance with these findings and its proposed role as on oncogene, knockout of CXCR5 in the Eμ-TCL1 CLL mouse model resulted in significantly delayed disease onset and disrupted leukemia cell migration [95].

CCR7 is another chemokine receptor that is upregulated in CLL [3, 61, 71, 96] and is believed to play a role in enabling cell entry into lymph nodes [97]. Higher mRNA and protein expression of CCR7 has been observed in unmutated compared to mutated IGHV CLL [46] and increased expression has been correlated with disease stage [76] and lymphadenopathy [46]. ZAP-70 has been shown to upregulate CCR7 via an ERK1/2- dependent mechanism that is also believed to regulate CXCR4 and CXCR5 [98]. The atypical chemoattractant GPCR CCRL2 (also known as CRAM) surface protein is expressed on CLL cells and is believed to regulate CCR7- and CCL19-mediated cell responses [99]. Finally, CCR7 and CXCR4 function together to upregulate matrix metallopeptidase 9 (MMP-9) to enable CLL cell migration [100]. Treatment of CLL patients with anti-CCR7 monoclonal antibody has shown promise in mice and humans [101].

Many chemokine receptors have varied expression in CLL based on the subtype of CLL being studied and the method used to identify the receptor. Similar to CXCR5, CCR6 protein expression was significantly reduced in poor-prognosis del 17p/11q CLL compared to normal CLL [93]. In addition, gene expression profiling showed that CCR6 was downregulated compared to memory B cells [61] while studies have reported a range from 23-100% of CLL/SLL cases that were positive for CCR6 [3, 10, 72, 102].

CX3CR1 protein was found to be present in 27-100% of CLL/SLL cases [103, 104] and CX3CR1 gene expression was higher in IGHV mutated than unmutated patients [105]. Interestingly, binding of CX3CL1 to CX3CR1 resulted increased CXCR4 MFI on CLL cells in vitro [104].

CCR1 and CXCR2 have both been detected in CLL/SLL. 4/35 (11%) cases of CLL/SLL were positive ACCEPTEDfor CCR1 via an IHC analysis MANUSCRIPT [14] and 9/13 CLL patient samples were positive for CCR1 as measured by flow cytometry [72]. Conversely, CXCR2 was detected in 7/51 (14%) CLL cases via IHC [106] but 0/13 (0%) cases via flow cytometry [72].

4. Mantle Cell Lymphoma Mantle cell lymphoma (MCL) arises from B cells located in the mantle cell zone surrounding the germinal center in secondary lymph nodes. MCL is traditionally

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characterized by overexpression of the oncogene cyclin D1 (CCND1), which frequently results due to a t(11;14) reciprocal translocation.

Among Non-Hodgkin’s lymphoma (NHL) subtypes, the endocannabinoid system has been most extensively described in MCL due to the consistent overexpression of the cannabinoid receptors CNR1 and CNR2 in malignant tissue compared to normal B lymphocytes [51, 107-112]. In a study of 107 MCL tissue samples, CNR1 and CNR2 mRNA was overexpressed in 98% and 100% of samples, respectively, and the degree of overexpression informed clinical factors [113]. CNR1 was significantly more strongly expressed in conventional MCL compared to indolent MCL [114] and significant associations were found between low CNR1 expression and lymphocytosis and leukocytosis as well as between high CNR2 expression and anemia [113]. Gene expression profiling of MCL primary tissue and cell lines compared to normal B cells found upregulation of CNR1 and the adrenergic receptor ADRB2 along with downregulation of the chemokine receptors CXCR5 and CCR6 and purinergic receptor P2RY11 in MCL although further flow cytometry found variable expression of the chemokine receptors [115, 116]. Therapeutic efforts to treat MCL primary cells and cell lines with cannabinoids resulted in decreased cell viability along with upregulated ceramide synthesis, apoptosis and cell death in MCL but not normal cells [107, 111, 112].

Another receptor that is highly expressed in MCL compared to other NHLs is the S1P receptor S1PR1. S1PR1 was detected via immunohistochemistry in 19/19 lymph node, 10/10 gastrointestinal tract and 9/9 bone marrow MCL cases but less frequently in other NHLs [117]. S1PR1 protein was also more highly expressed than S1PR2 and S1PR3 in MCL [118]. Finally, exome sequencing of patient samples identified mutations in the S1PR1 gene in 2/11 (18%) cases [119] as well as in the Mas-related GPCR MRGPRF in 3/56 (5%) cases [120].

The estrogen receptor GPER1 is enriched in MCL as it was detected via IHC in 94/157 (60%) MCL patient samples compared to 1/20 (5%) FL cases and 6/20 (30%) diffuse large B cell lymphoma (DLBCL) cases. Knockdown of GPER1 in MCL cell lines resulted in stabilization of microtubules, inhibition of proliferation, and activation of apoptotic pathways and combining GPER1 inhibitors with additional chemotherapeutics had a synergistic effect [121].

Chemokine receptor expression in MCL is shown in Table 1. CXCR4, CXCR5 and CCR6 protein was detected in the majority of patients [10, 72, 102, 118] and CX3CR1 was found to beACCEPTED present in up to 75% of cases MANUSCRIPT depending on the method used to identify the receptor [103]. CCR7 was more highly expressed in MCL than normal B cells [71] and its protein expression varied based on anatomical site [118]. CCR1, CCR3 and CXCR3 were also detected in a small percentage of patients [4, 14, 68, 72].

5. Burkitt Lymphoma Burkitt lymphoma, follicular lymphoma and the germinal center subtype of DLBCL (GCB DLBCL) are all believed to arise from germinal center B cells. The germinal center forms in response to foreign antigen presentation in order to produce specific

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antibodies against the antigen. B cells rapidly proliferate within the germinal center and pass through multiple rounds of selection to identify cells producing highly specific antibodies. B cells that successfully produce high-quality antibodies are retained while the remaining cells succumb to apoptosis.

Germinal center B cells rely on expression of chemokine receptors CXCR4 and CXCR5 to move between the dark and light zone of the germinal center where proliferation and selection occur, respectively. CXCR5 was discovered in BL and was originally named Burkitt Lymphoma Receptor 1 (BLR1) [122]. Flow cytometry on a small number of samples found that CXCR5 was well expressed in BL patient samples while CXCR4 was expressed in some cases [2]. Multiple CXCR4 inhibitors have shown success inducing cell death in BL cell lines including BKT140 [123], plerixafor and GEZ-644494 [124] and palmitoylated peptides called pepducins [125]. Interestingly, treatment with the CD20 antibody rituximab resulted in increased surface expression of CXCR4 whereas treatment with Shiga-like toxin reduced the amount of CXCR4 available [126].

The prostaglandin E receptors PTGER1, PTGER2, PTGER3 and PTGER4 are believed to play a proinflammatory role in BL [127]. In particular, PTGER4 has been identified on a variety of B cell lymphoblast cell lines, including BL, and is thought to inactivate NF- κB to sensitize cells to chemotherapeutics and induce apoptosis [128]. Other GPCRs which have been identified in BL include the cannabinoid receptor CNR1, which demonstrated strong and uniform staining via IHC in a BL patient sample [51], and the trace amine associated receptor TAAR1 protein, which was detected by Western blot in normal B cells along with a variety of B cell lymphoma cell lines including BL. Treatment of the BL cell line L3055 with a TAAR1 agonist was toxic to the cells via an apoptotic mechanism [129].

6. Follicular Lymphoma Follicular lymphoma encompasses a range of lymphomas emerging from the germinal center B cell follicle that can vary in presentation from indolent to aggressive disease. FL can transform from a small cell to a large cell disease and this transformation is associated with poor clinical outcome.

Next generation sequencing technologies have revolutionized the identification of genetic aberrations in NHL and have revealed multiple mutations in GPCRs in FL. Whole exome sequencing identified loss of function mutations in CXCR4 and P2RY8 [130] as well as copy number loss of CXCR4, P2RY8 and S1PR2 in FL [131]. Mutation or copy number loss of these receptorsACCEPTED has also been noted MANUSCRIPTas significantly more common in transformed FL compared to non-transformed FL [131, 132]. These studies suggest a tumor suppressive role for these receptors. CXCR4 protein is well-expressed in FL [133] but S1PR2 surface expression is not as highly expressed compared to other NHLs [118]. P2RY8 has also been noted for a fusion to the SOX5 transcription factor in primary splenic FL [134] .

Gene expression profiling of FL cases revealed an increase in the GPCRs PTGER4, CX3CR1, GPR183 (also known as Epstein-Barr virus-induced GPCR 2; EBI2) and

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CNR1 compared to normal tonsillar B cells [103]. Further investigation into the role of cannabinoid receptors in FL found that CNR1 and CNR2 mRNA were both upregulated compared to reactive lymph node tissue but that their expression did not correlate to tumor grade [51]. In addition, CNR2 transcript expression was found to be anatomically controlled as it was significantly upregulated in duodenal as compared to nodal FL [135].

The expression of chemokine receptors in FL is summarized in Table 1. IHC and flow cytometry of patient samples found that CXCR5 is well-expressed in FL [3, 10, 71, 72, 118] and SNPs in CXCR5 were associated with increased risk of FL and subsequent survival [136, 137]. FL cells are known to produce the ligand for CXCR5, CXCL13, and it is believed they express CXCR5 to respond to CXCL13 and form the architecture of lymphoid follicles [138]. Monoclonal antibodies targeting CXCR5 were found to be reactive against primary cutaneous FL but not closely related lymphomas [139] and have been shown to inhibit tumor growth in mouse models [140]. A trifunctional bispecific antibody targeting CXCR5::CD3 suppressed tumor growth and increased survival in a xenograft model of B cell lymphoma by recruiting T cells to CXCR5-expressing B cells and co-stimulating the activation of CD4+ and CD8+ T cells to lyse B cells [140].

CXCR5 is not only critical in the B cell lineage in FL. A high proportion of CXCR5- expressing follicular helper T cells (TFH) were observed in FL but not in closely related DLBCL [141] and further investigation revealed that FL regulatory T cells (Tregs) used a CXCL13-CXCR5 autocrine loop for positioning and upregulation of additional chemokine receptors [142]. There was also an increase in the number of CXCR5- expressing memory T cells along with changes in specific CXCR5+ memory T cell subsets in particular [143]. Finally, gene expression profiling showed that as a cell transitioned from normal lymph node to reactive lymph node to FL, CXCR5 expression increased in regulatory T cells whereas S1PR1 and CCR7 decreased [142].

While CXCR5 promotes migration towards CXCL13, CXCR4 mediates migration towards CXCL12-producing stromal cells [144] and is expressed on the surface of normal, relapse and transformed FL cells [3, 10, 71, 72, 118, 131, 133, 145]. Multiple therapeutic possibilities involving CXCR4 have been attempted including monoclonal antibodies [139], the pan-PI3K inhibitor BKM120 [146] and crosslinking of LLT1 [147].

CCR1 expression is highly enriched in FL compared to other lymphoid malignancies [14]. Although increased CCR1 mRNA expression was found to be associated with worse outcome in a one study [148], CCR1 protein expression was not correlated with overall survivalACCEPTED or disease stage in a larger MANUSCRIPTstudy [14].

Presentation of FL can vary in anatomical location and multiple chemokine receptors have expression patterns specific to anatomic subtype. CCR9 was found to be significantly more immunopositive in gastrointestinal FL compared to nodal FL and its presence in nodal FL was found to be an indicator for future gastrointestinal involvement [149]. Similarly, CCR6 was expressed in 28/32 (88%) duodenal FL patients as compared to only 14/27 (52%) nodal follicular lymphoma patients via immunohistochemistry [135] although a separate study did not observe CCR6 in any of 17 patient samples [102].

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CX3CR1 was identified in 25-80% of cases depending on the method used to identify the receptor [103] and both CX3CR1 and CCR3 mRNA expression were significantly decreased in FL compared to reactive follicular hyperplasia [150]. Individual studies have also concluded that CCR2 protein is expressed in FL [151] whereas CCR7 [118] and CXCR3 are not as highly expressed [4].

7. Diffuse Large B Cell Lymphoma DLBCL is a clinically and molecularly heterogeneous disease comprised of two major subgroups that can be identified through gene expression profiling: activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL. DLBCL can originate at multiple anatomical sites and GPCR expression can vary between DLBCL subtype and disease location.

The S1P receptor family plays a prominent role in DLBCL. S1PR2 acts as a tumor suppressor in DLBCL and high gene expression of S1PR2 has been identified as a prognostic factor indicating increased likelihood of survival [152]. Sequencing of 106 DLBCL patient samples and cell lines found 28/106 (26%) had a mutation in the 5’ region of the S1PR2 gene within the intron between exons 1 and 2 [153]. Mutations have also been identified in the coding region of the gene and are suspected to be a result of aberrant somatic hypermutation [153-155]. In an effort to confirm the role of S1PR2 as a driver mutation in DLBCL, knockout of S1PR2 in mice were found to develop spontaneous disease resembling DLBCL [153]. In addition to mutations in S1PR2, polymorphisms in CCR8 were found to be associated with DLBCL and SNPs in CXCR5 were associated with increased DLBCL risk in the GCB subgroup in particular [136].

IHC of tumor samples found that S1PR1 was expressed on 54-58% of primary testicular DLBCL and 13-40% DLBCL not otherwise specified (DLBCL-NOS) cases [156, 157]. S1PR1 expression was an independent indicator of poor outcome in patients with stage I and II DLBCL [156] and for rituximab-treated patients [157]. S1PR1 was shown to colocalize with phospho-STAT3 in ABC patient samples and treatment of mice with lymphoma with the S1P antagonist FTY720 inhibited Stat3 activity and reduced tumor growth [158]. Similarly, knockout of S1PR1 in cell lines reduced tumor growth and invasion after transplant into mice [158].

Gene expression profiling with corresponding clinical data supported the notion that increased expression of S1PR1 in DLBCL was associated with poor outcome [159-161]. These studies ACCEPTEDalso identified increased expression MANUSCRIPT of the GPCRs GPR183, CCR7, ADRB2 and CNR2 as risk factors for poor outcome [161]. However, a conflicting study found that CNR2 protein expression was not associated with outcome or related to ABC/GCB subtype [162]. While the significance of cannabinoid receptor expression in DLBCL remains to be elucidated, it has been shown that CNR1 and CNR2 mRNA were both upregulated in DLBCL compared to reactive lymph node tissue [51] and that 45/79 (57%) patients were CNR2 immunopositive [162].

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Chemokine receptor expression in DLBCL is shown in Table 1. Consistent with its role in NHL, CXCR4 plays an important role in enabling cell migration in DLBCL. Increased CXCR4 expression was associated with worse survival in both ABC and GCB subtypes for 468 patients treated with the standard therapy R-CHOP [163] and a separate study of 94 patients found that those positive for CXCR4 has reduced survival and increased recurrence of disease [164]. However, a smaller cohort of 70 Korean patients did not identify an association between CXCR4 expression and survival [165]. At the cellular level, strong nuclear CXCR4 staining was correlated with systemic DLBCL whereas strong cytoplasmic CXCR5 staining was correlated with CNS involvement [166]. Furthermore, hypoxia was associated with upregulation of CXCR4 protein [167] and such an increase in CXCR4 expression is believed to increase cell dissemination [164]. IHC revealed that 80% of patients had CXCR4 coexpressed with NF-κB [165], which is often mutated in DLBCL, while CXCR4 itself contains an AIDCA somatic hypermutation hotspot that is suspect to mutation [168]. Various therapeutics targeting CXCR4 in DLBCL have been studied including the CXCR4 antagonist plerixafor which enhanced rituximab treatment [169], BTK140 which inhibited growth in cell lines [163], PIM inhibitors which impaired proliferation and CXCR4-mediated migration [170], and in vivo blocking of CXCR4 which was critical for regulatory T cell attraction to lymphoma [171].

CCR1 expression was IHC positive in 77/209 (37%) of DLBCL cases and associated with the ABC subtype of DLBCL but did not correlate with survival [14]. At least 75% of gastric extranodal DLBCL cases had positive mRNA expression for CCR1 along with the chemokine receptors CCR5, CCR7, CCR8, CCR9, CXCR3, CXCR5, CXCR6, CXCR7 and XCR1 [172, 173]. IHC was also positive in the majority of extranodal cases for CCR8, CCR9, CXCR4, CXCR6 and CXCR7 [173].

Multiple reports have highlighted the difference in chemokine receptor expression based on anatomical location of DLBCL. Similar to FL, IHC staining of CCR9 was stronger in gastrointestinal DLCBL compared to nodal DLBCL [149]. XCR1 surface protein was found to be significantly elevated in DLBCL cases manifesting in the bone marrow [174] and also present in the majority of extranodal DLBCL cases [173]. CXCR3 protein expression was higher in thyroid DLBCL than stomach DLBCL [175] and has also been observed in case reports of epidermotropic B cell lymphoma [176] and intravascular large B cell lymphoma [177]. Closely related primary mediastinal large B-cell lymphomas (PMBCLs or MLBCLs) have been characterized by increased CCR9 and decreased CCR6, CCR7 and CXCR5 immunoreactivity compared to nonmediastinal DLBCL [14, 178]. Finally, ACCEPTEDalthough not a chemokine receptor, MANUSCRIPT analysis of 57 NHL biopsies found that 9/10 (90%) PMBCL and 1/9 (11%) DLBCL cases were positive for the leukotriene receptor CYSLT1 but no positive samples were found in other NHL subtypes [179].

Other chemokine receptors that have been observed in DLBCL include CX3CR1, which was found to be present in up to 43% of cases and enriched in the GCB subtype [103]. IHC of 80 DLBCL cases found that 10 (12.5%) were positive for CCR4 expression but this had no correlation with patient outcome [180]. CCR4 expression may be subtype specific as DLBCL originating from the thyroid and stomach had very few cells positive

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for CCR4 protein expression [175] but multiple case reports of CCR4 in other DLBCL subgroups have been reported [180-182].

8. Marginal Zone Lymphoma Marginal zone lymphoma (MZL) comprises three lymphoma entities that arise from the marginal zone surrounding the germinal center: extranodal MZL or mucosa-associated lymphoid tissue (MALT), splenic MZL, and nodal MZL. Deep sequencing of nodal MZL patients found that 5/35 (14%) cases had a mutation in the adhesion receptor ADGRV1 (also known as GPR98) and the cadherin receptors CELSR1, CELSR2 and CELSR3 were each mutated in 2/35 (6%) cases [183]. In addition to mutation, t(X;14) translocations causing upregulation of purinergic receptor GPR34 were observed in 2/61 (3%) cases of MALT, 1/43 (2%) cases of nodal MZL and 1/19 (5%) cases of extranodal DLBCL [184]. When compared to healthy B and T cells, GPR34 mRNA expression was significantly upregulated in MALT, nodal and splenic MZL and increased gene expression of GPR34 in was correlated with high expression of the orphan receptor GPR82. Overexpression of GPR34 in cell culture experiments resulted in increased cell proliferation and increased phosphorylation of the kinases ERK and PKC and cAMP response element-binding protein (CREB) [185].

Other GPCRs that had elevated expression in MZL include the S1P family receptors S1PR1 and S1PR3 [118] and the gastrin/cholecystokinin receptor CCKBR [186]. A fraction of MALT patients were immunopositive for the somatostatin receptors SSTR1 (1/55, 2%), SSTR2 (15/55, 27%), SSTR3 (20/55, 36%), SSTR4 (10/55, 18%) and SSTR5 (28/55, 50%) and there was a significant association between SSTR5 negativity and poor patient outcome [187]. SSTR-receptor scintigraphy was demonstrated to be useful for staging and follow-up in MALT [188] although there is disagreement concerning whether gastric tumors have higher SSTR3, SSTR4 and SSTR5 than extragastric tumors [187, 188].

Chemokine receptor expression in MZL subtypes is shown in Table 1. CXCR3 is strongly expressed on the surface of the majority of MZLs and was identified via IHC in 14/14 (100%) patients with splenic MZL, 15/16 (94%) patients with extranodal MZL [4] and 5/5 (100%) cases of epidermotropic MZL [176]. One study found that only 13% of cutaneous MZLs were CXCR3-positive compared to 85% of other extranodal MZLs implying that CXCR3-negative patients comprise a unique subtype of MZL [189]. CXCR3 is a predictor of non-responsiveness for H. pylori eradication therapy implying that CXCR3 helps protect tumors from this treatment [190]. MALT lymphoma cells are known to expressACCEPTED CXCR3 and migrate to theMANUSCRIPT CXCR3 ligand CXCL9 (also known as MIG) [191] and both high- and low-grade MALT originating from the thyroid and stomach had high CXCR3 and low CCR4 cell surface expression [175]. However, CCR4 protein expression itself was significantly more highly expressed in trisomy 3-positive compared to trisomy 3-negative MALT and correlated with advanced disease stage and poor prognosis [172].

Although CXCR4 and CXCR5 are generally expressed in MZL [72, 118, 172, 173], flow cytometry of splenic MZL tissues with minimal lymphadenopathy had significantly

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reduced expression of CXCR4, CXCR5 and CCR7 compared to normal B cells and B cell neoplasm with nodular dissemination such as CLL, MCL and FL [71]. Exome sequencing found 2/35 (6%) cases of MZL had a mutation in CXCR4 [183] and the CXCR4WHIM mutation was found in 1/20 (5%) MZL patients [192]. A significant correlation has been observed between CXCR4 expression and bone marrow involvement and loss of CXCR4 expression combined with upregulation of CXCR7 has been suggested to correlate with MALT progression to DLBCL [173].

Other chemokine receptors that are expressed in MZL include CCR8, CCR9, CXCR6 and CXCR7, which were immunopositive in the majority of gastric MALT lymphomas [173]. Two independent studies found that 84-88% of MALT cases stained positive for CCR6, which is known to play a role in migration and B cell maturation [102, 135]. In addition, CCR1 protein expression was found to be subtype-specific as 3/6 (50%) nodal MZL cases expressed CCR1 compared to only 1/21 (5%) extranodal MZL and 0/5 splenic MZL cases [14].

9. Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia: Lymphoplasmacytic lymphoma (LPL) and its subgroup Waldenstrom macroglobulinemia (WM) are rare and indolent lymphomas that arise from fully differentiated B cells. The only family of GPCRs that has been well studied in LPL/WM is the chemokine receptor family. Flow cytometry of WM cell lines and patient samples found 13 CXC and CC chemokine receptors were expressed to some degree [4, 14, 193] and CXCR4, in particular, was highly expressed and found to be necessary for WM migration to bone marrow [193]. CXCR4 was shown to interact with integrin α4β1 (also known as Very Late Antigen-4; VLA-4) to regulate homing and adhesion, and treatment of WM cell lines with a CXCR4 antagonist inhibited migration and signaling [193, 194].

Driver mutations in CXCR4 are an important determinant of clinical phenotype and survival in WM/LPL [195] and the role of CXCR4 in homing, migration and dissemination of WM/LPL has been well reviewed [196]. Mutations in CXCR4 were detected in 17/47 (36%) LPL cases and mutation correlated with increased bone marrow infiltration and lower leucocyte, hemoglobin and platelet counts [197]. CXCR4 mutations were also identified in 24-27% of WM patients and the mutations were heterozygous and located in the carboxy-terminal tail [198-200]. The CXCR4 C1013G mutation was detected in 37/131 (28%) cases of LPL and a novel anti-CXCR4 antibody was shown to affect survival and apoptosis signaling in WM cells [201]. Separating LPL/WM into subgroups revealed CXCR4WHIM mutations in 2/12 (17%) Immunoglobulin M monoclonal gammopathyACCEPTED of undetermined MANUSCRIPTsignificance (IgM MGUS), 0/7 (0%) Non-IgM MGUS, 44/102 (43%) untreated WM and 21/62 (34%) treated WM [192].

CXCR4 cell surface expression was increased in CXCR4-mutated cases of WM compared to CXCR4-wild type [198] but overall CXCR4 was found to be lower in WM than other NHLs [202]. Nearly all cases that have CXCR4 mutations also have MYD88 mutations [197, 200], however a small study found that only about 29% of patients with MYD88 mutations in WM also have a CXCR4 mutation [203]. Frameshift mutations in

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CXCR4 have also been identified and shown to result in receptor internalization in response to CXCL12 [204].

10. Hairy Cell Leukemia: Hairy cell leukemia (HCL) is a rare disease that accounts for approximately 2% of leukemias and is typically defined by cells that contain the BRAF kinase p.V600E mutation [205]. Gene expression profiling of HCL has suggested that these tumors arise from a memory B cell of origin and that transformation to lymphoma involves changes in expression of chemokine and adhesion receptors [206]. Similar to LPL/WM, studies of GPCRs in HCL mainly concern chemokine receptors and these findings are summarized in Table 1. At least two independent reports have concluded that CX3CR1, CXCR3. CXCR4, CXCR5, CCR5 and CCR6 have been identified in patient cases [3, 4, 10, 72, 103]. Meanwhile, cell surface expression of CCR7, CXCR4 and CXCR5 was found to be significantly lower in HCL compared to normal B cells or B cell neoplasms with nodular dissemination [3, 71, 206].

11. Conclusions GPCRs represent an important component of B cell signaling and play critical roles in cell migration, proliferation, apoptosis, development, and function. Genetic events that change expression or function of GPCRs, such as mutations, fusions or copy number alterations, have significant consequences and can serve as driver events in B cell malignancies. Although many of these events have been described, significant research remains to elucidate the full expression panel of GPCRs during all stages of B cell development and their function in the normal immune system and oncogenesis. Deciphering these elements will lead to an increase in therapeutics targeting these receptors and ultimately improve patient outcomes.

Funding Sources: This research was supported by the Intramural Research Programs of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health.

Figure Legends: Figure 1. GPCRs in B cell lymphoma. GPCRs that are upregulated (green arrow), downregulated (red arrow), mutated (blue X) or are known to significantly inform prognosis (purple star) are indicated for individual lymphomas. The cell of origin that gives rise to each type of lymphoma is shown in the context of normal B cell development. ACCEPTED MANUSCRIPT Table 1. Chemokine receptor expression in B cell lymphomas. The proportion of tumor samples that were reported as positive for individual chemokine receptors are shown across thirteen NHL subtypes. Data were grouped according to the method used to identify the receptor (IHC = immunohistochemistry; FC = flow cytometry; mRNA = quantitative PCR). Blank cells indicate that measurements have not been reported. Cell color reflects the total percentage of positive tumor cases (grey = 0%; lightest blue = 1- 24.9%; medium blue = 25-49.9%; darker blue = 50-74.9%; darkest blue = 75-100%). * indicates significant differences in expression exist among subgroups within the

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particular disease. Data were assembled from the following references: [2][3][4][10][14][68][72][76][102][106][135][151][163][164][165][172][173][175][178][ 189][190][191].

Table 2. GPCRs in B cell lymphoma studies. GPCRs that have been investigated in B cell lymphoma are shown corresponding to the specific diseases in which each receptor has been studied.

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202. Mancuso, P., et al., If it is in the marrow, is it also in the blood? An analysis of 1,000 paired samples from patients with B-cell non-Hodgkin lymphoma. BMC cancer, 2010. 10(1): p. 644. 203. Ballester, L.Y., et al., Clinical Validation of a CXCR4 Mutation Screening Assay for Waldenstrom Macroglobulinemia. Clinical Lymphoma Myeloma and Leukemia, 2016. 16(7): p. 395-403. e1. 204. Cao, Y., et al., CXCR4 WHIM‐like frameshift and nonsense mutations promote ibrutinib resistance but do not supplant MYD88L265P‐directed survival signalling in Waldenström macroglobulinaemia cells. British journal of haematology, 2015. 168(5): p. 701-707. 205. Grever, M.R., J.S. Blachly, and L.A. Andritsos, Hairy cell leukemia: Update on molecular profiling and therapeutic advances. Blood Rev, 2014. 28(5): p. 197- 203. 206. Basso, K., et al., Gene expression profiling of hairy cell leukemia reveals a phenotype related to memory B cells with altered expression of chemokine and adhesion receptors. Journal of Experimental Medicine, 2004. 199(1): p. 59-68.

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Figure 1

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Table 1. The fraction of tumor samples that were reported as positive for individual chemokine receptors are shown across thirteen NHL subtypes. Data were grouped according to the method used to identify the receptor (IHC = immunohistochemistry; FC = flow cytometry; mRNA = quantitative PCR). Blank cells indicate that measurements have not been reported. Cell color reflects the total percentage of positive tumor cases (grey = 0%; lightest blue = 1-24.9%; medium blue = 25-49.9%; darker blue = 50-74.9%; darkest blue = 75-100%). * indicates significant differences in expression exist among subgroups within the particular disease. Data were assembled from the following references: [2][3][4][10][14][68][72][76][102][106][135][151][163][164][165][172][173][175][178][189][190][191].

Pro B- Early Pre Pre B- GPCR Method B-ALL CLL SLL MCL BL FL DLBCL MZL MALT HCL ALL B-ALL ALL

CCR1 IHC 1/17 0/4 122/349 85/217 3/11* 12/32 0/4

FC 0/13 0/17 0/12 0/9 9/13 0/6 0/5 0/4 0/5 0/5 4/9

mRNA 18/18 24/26

CCR2 IHC

FC 0/13 0/17 0/12 0/9 6/58 1/6 3/5 0/4 8/11 0/5 6/9

mRNA 6/6

CCR3 IHC

FC 5/8 6/17 6/12 3/5 13/13 1/6 2/5 0/4 0/5 0/5 7/9

mRNA

CCR4 IHC 11/91 7/19

FC 7/11 8/17 6/12 3/5 0/45 0/4

mRNA

ACCEPTED MANUSCRIPT CCR5 IHC 8/8 16/17

FC 0/13 0/17 0/32 0/9 1/81 3/15 5/9 0/4 4/24 3/5 16/21

mRNA 18/18 24/26

CCR6 IHC 7/11 42/76* 19/19

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FC 0/13 0/17 0/31 0/9 29/52 10/10 12/12 0/4 4/24 5/5 21/21

mRNA 8/8 16/16

CCR7 IHC 3/15

FC 4/11 6/17 5/12 4/5 21/21 4/8 0/4 1/13 0/4

mRNA 16/18 23/26

CXCR1 IHC

FC 0/8 5/17 0/12 3/5 3/58 3/6 0/5 0/4 2/5 2/5 4/9

mRNA

CXCR2 IHC 7/51

FC 4/7 15/15 12/12 5/5 0/58 4/6 0/5 4/4 2/5 2/5 2/9

mRNA

CXCR3 IHC 3/9 1/30 1/27 9/11 36/67* 77/127* 5/7

FC 8/10 15/15 19/19 5/5 62/65 13/15 4/14 4/4 15/22 5/5 15/29

mRNA 16/18 20/26

CXCR4 IHC 255/505 2/5

FC 11/13 8/17 8/12 3/5 57/57 15/15 9/9 2/4 24/24 5/5 21/21

mRNA 7/20 15/26

CXCR5 IHC 13/15

FC 3/9 5/17 4/12 13/13 113/115 12/15 10/10 4/4 26/26 6/7 18/22

mRNA 17/18 23/26

ACCEPTED Color MANUSCRIPT25 - 50 - 75 - 0% 1-24.9% scale: 49.9% 74.9% 100%

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Table 2. Publication references that describe GPCR expression or function in NHL are shown for each individual disease and GPCR.

GPCR B-ALL CLL/SLL MCL BL FL DLBCL MZL/MALT LPL/WM HCL

ACKR3 [13] [80] [173] [173]

ADGRV1 [183]

ADORA2A [55]

ADORA2B [55]

ADRB2 [52][53] [116] [161]

CALCR [56]

CCKBR [186] [14][72] [2][14][1 [14][72][172][ CCR1 [2][14] [14][72] [14][72][116] [116][1 [14][172][173] [14][193] [14][72] 16] 173] 48] [72][15 CCR2 [2] [72] [72] [2] [72] [193] [72] 1] [72][11 CCR3 [2] [72] [72][116] [2][116] [72] [193] [72] 6][150]

CCR4 [2][12] [12] [2] [175][180-182] [172][175] [193] [2][3][1 [3][10][ [3][10][ CCR5 [3][10][72]ACCEPTED [10][72] [2] MANUSCRIPT[172][173] [72][172][173] [193] 0] 72] 72] [2][3][1 [3][10][61] [10][72][102] [3][10][ [72][102][135] [3][10][ CCR6 [2][116] [172][178] [193] 0] [72][93][1 [115][116][1 72][102 [172] 72]

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02] 18] ][116]

[3][12][43] [3][71][ [2][3][1 [46][61][7 [71][116][11 [118][161][166][ [71][118][172] [3][71][ CCR7 [2][116] 116][11 [193] 2][94] 1][76][94][ 8] 172][173][178] [173] 206] 8][142] 96-101]

CCR8 [136][172][173] [172][173] [193]

CCR9 [12] [12] [149] [149][173][178] [173]

CCR10 [172] [172]

CCRL2 [99]

CELSR1 [183]

CELSR2 [183]

CELSR3 [183] [51][107- [51][10 CNR1 [51] [50][51] [51] [51] [51] 116] 3] [51][107- [51][13 CNR2 [51] [50][51] [51] [51][161-162] [51] 116] 5] [103][1 CX3CR1 [103-105] [103] [103] [103] ACCEPTED MANUSCRIPT50]

CXCR1 [2] [72] [72] [72] [172] [72][172] [193] [72]

CXCR2 [2] [72][106] [72] [72] [72] [193] [72]

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[3][4][12][ [4][72][172][1 [2][3][4 66][68- [3][4][7 [4][172][173][17 [3][4][6 CXCR3 [4][68][72] [4] 73][175- [193] ][12] 70][72][76 2] 5] 8][72] 177][189-191] ] [3][10][ [3][10][46] 71][72][ [71- 118][12 [2][123- [118][132][139][ [71][72][118][ [2][3][5 89][91][92 [10][71][72][ 3][131- [3][10][ CXCR4 126][132 163- 139][172][173] [192-204] -11] ][100][125 102][118] 133][13 71][72] ] 173][178][202] [183][192] ][192][202 9][144- ] 147][15 1][202] [3][4][1 [3][4][10][ [4][10][71][7 0][71][7 [2][3][4 [118][136][139][ [4][71][72][11 [3][4][1 12][71][72 2][90][115][1 [2][4][11 2][90][1 CXCR5 ][10][1 141][166][172][ 8][137][172][1 [4][193] 0][71][7 ][90- 16][118][137 6][122] 16][118 2] 173][178] 73] 2] 95][137] ] ][136- 142]

CXCR6 [12] [12] [172][173] [173]

CYSLT1 [57][179] [179] [179] [179] [179] [179]

EDNRA [58]ACCEPTED MANUSCRIPT

F2R [39] [39]

FZD3 [65][66]

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FZD6 [63][64]

GPER1 [11] [121] [121] [121]

GPR34 [38] [184] [184][185]

GPR65 [59]

GPR82 [185] [32][33 GPR132 ]

GPR183 [103] [161]

LPAR1 [47-49]

LPAR3 [47]

LPAR5 [47]

LTB4R [60]

MRGPRF [120]

NTSR1 [62]

NTSR2 [62] [130- [1][15- P2RY8 132][13 31] ACCEPTED MANUSCRIPT 4]

P2RY11 [55] [115][116]

PTGER1 [37] [127]

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PTGER2 [34-37] [127]

PTGER3 [37] [127] [127][12 PTGER4 [37] [103] 8] [117][1 [40][11 [40][43- [117][118][156- S1PR1 [117-119] [117] 18][142 [117][118] [117] 7] 46][117] 161] ] [118][1 [118][136][152- S1PR2 [40] [40][43] [118] 31][132 [118] 155] ]

S1PR3 [118] [118] [118] [118]

S1PR4 [40] [40] SSTR1- [187][188] SSTR5

TAAR1 [129] [129] [129] [129]

TACR1 [41] [41]

TBXA2R [61]

XCR1 [172-174] [172][173] ACCEPTED MANUSCRIPT

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Highlights  GPCRs regulate B cell migration, proliferation, apoptosis, development and function  Mutation, fusion and copy number alterations of GPCRs occur in B cell malignancies  B cell lymphoma subtypes contain unique GPCR expression and aberration patterns  GPCR mutations and changes in expression can serve as biomarkers of lymphoma  Better understanding of GPCRs in lymphoma will improve clinical outcomes

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