Circulating biomarkers in classical Hodgkin lymphoma
Plattel, Wouter Johannes
DOI:
10.33612/diss.97631424
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2019
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Plattel, W. J. (2019). Circulating biomarkers in classical Hodgkin lymphoma. Rijksuniversiteit Groningen.
https://doi.org/10.33612/diss.97631424
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CHAPTER 5
The role of microRNAs in Hodgkin lymphoma
Wouter J Plattel, Joost Kluiver, Arjan Diepstra, Lydia Visser, Anke van den Berg
Abstract
In this chapter we summarize the current knowledge on microRNA (miRNA) expression, function and putative clinical value in Hodgkin lymphoma (HL). HL is the second most common lymphoma subtype and mainly affects young adults or elderly. Malignant cells in HL are scarce, usually less than 1%, and these cells are of B cell origin. As miRNAs are involved in almost all known biological processes, deregulation of miRNAs is thought to play an important role in the pathogenesis of HL. MiRNA expression studies have been performed on HL cell lines, laser microdissected tumor cells and total tumor tissue sections. Despite the use of different tissue specimens, a reasonable overlap has been found in the differentially expressed and highly abundant miRNAs. The most consistent highly expressed miRNAs include several known cancer related miRNAs, e.g. miR-16, miR-20a, miR-21 and miR-155. Functional studies on (deregulated) miRNAs in HL are performed for a limited number of miRNAs, but multiple functional target genes have been identified. Inhibition or overexpression of individual miRNAs in HL resulted in altered expression of target genes that are involved in apoptosis, proliferation, cytokine production or plasma cell differentiation. In addition, low miR-135a levels in HL tumor cells are associated with low disease free survival and high relapse rates. These miRNAs are potential novel therapeutic targets or prognostic markers. However, more studies are needed to identify possible clinical applications for miRNAs in HL.
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Introduction
Hodgkin lymphoma (HL) is the second most common lymphoma subtype with an incidence of about 9,100 and 18,000 new HL patients per year in the US and Europe respectively. About 50% of the HL patients are diagnosed between 15 to 34 years of age, but the disease can occur at any age. In western countries a bimodal age-incidence distribution is observed, but there is a considerable variation in incidence and age distribution in different parts of the world. The tumor cells of HL are in almost all cases of B cell origin, although some rare cases of T cell type HL have been reported. HL is a very peculiar lymphoma subtype since it consists of a minority of neoplastic cells that generally comprise less than 1% of the total cell population in a background of reactive cells.1 The vast majority of the HL patients present with the so-called
classical (c)HL subtype, which includes nodular sclerosis, lymphocyte rich, mixed cellularity and lymphocyte depleted subtypes. The cellular background consists of varying amounts of T and B cells, eosinophils, mast cells, macrophages and plasma cells and this composition determines the cHL subtype. Nodular lymphocyte predominant HL (NLPHL) is considered to be a different entity based on pathological and clinical features and accounts for only 5% of all HL cases. The tumor cells of cHL are referred to as Hodgkin Reed Sternberg (HRS) cells and the tumor cells in NLPHL are called lymphocyte predominant (LP) cells.
Approximately 85% of cHL patients are cured by risk adapted chemotherapy with or without radiotherapy, but treatment related late toxic effects like cardiovascular disease, solid malignancies and secondary leukemias can occur. NLPHL is a much more indolent disease than cHL and is therefore only treated with more intensive chemotherapeutic regimens in patients with locally advanced or advanced stage disease. Deaths in NLPHL are usually caused by treatment toxicity and occasional transformation to non-Hodgkin lymphoma.
The origin of the HRS cells in cHL has been controversial for a long time because the immunophenotype of these cells is strikingly different from other hematopoietic cells.2 Detection
of clonal immunoglobulin gene rearrangements, despite the lack of immunoglobulin protein production, confirmed a B cell origin.3 HRS cells frequently have a high number of immunoglobulin
(Ig) gene mutations leading to stop codons and other alterations that disable the gene in a proportion of the cases.4 These studies indicated that HRS cells originate from germinal center
B cells that lost the capacity to be selected by antigens. Such alterations are lethal to normal B cells, but HRS precursor cells are able to survive. At the time of diagnosis, HRS cells have virtually lost their B cell identity since they show no or strongly reduced expression of many common B cell markers (sIg, CD19, CD20, CD22, CD79a) and B-cell transcription factors (Bob-1, Oct-2, PU.1, Pax5).5,6 LP cells are also derived from germinal B cells, but these cells do have a functional
Infection with Epstein Barr virus (EBV) is found in 20-40% of cHL patients in the Western world.8
This infection is clonal and considered to be an early event that is essential for the malignant transformation of HRS precursor cells. In EBV- cHL the causative tumor initiating event is still unknown. Many candidate genes have been screened and mutations in known oncogenes, including TP53, FAS and IkBa, have been found in a variable number of cases in EBV+ and EBV- cHL patients. More recently, somatic mutations in the NF-κB suppressor gene, TNFAIP3, have been reported in 14 out of 20 (70%) of the EBV- cHL and in only 2 out of 16 (12.5%) of the EBV+ cHL.9 The high
mutation frequency in EBV- cHL might indicate that inactivation of the NF-κB suppressor gene plays an important role in cHL pathogenesis. EBV infection and loss of TNFAIP3 are two pathogenetic mechanisms that lead to constitutional activation of NF-κB, which is a hallmark of cHL.
I. miRNAs in Hodgkin lymphoma
Deregulation of miRNAs in hematopoietic cells has been linked directly to the development of several lymphoma subtypes. So far, both oncogenic, e.g. miR-17~92 and miR-21, and tumor suppressor miRNAs, e.g. miR-15a~16-1 and miR-150, have been reported in various hematological malignancies.10-18
Retrospectively, the first report on a miRNA in HL was published in 2003, when a high expression of the B cell integration cluster (BIC) gene was observed in HL cell lines by qRT-PCR.19 A tumor
cell specific expression pattern was confirmed by RNA-ISH in the HRS cells of primary HL cases. Expression of BIC was also shown in part of the germinal center B cells, and the transcripts were usually located in the nucleus in both HRS cells and germinal center B cells. The BIC gene locus was originally described as a commonly targeted region in an avian leukosis virus induced chicken model for B cell lymphoma.20 Overexpression of the BIC gene in chicken embryos resulted in the
induction of B cell lymphoma and enhanced the oncogenic potential of MYC.21 The BIC transcript
does not have a functional open reading frame, but does contain a highly conserved stem-loop like region. Cloning of small RNAs from mouse hematopoietic cells resulted in the identification of miR-155, which was derived from the conserved stem-loop region that was located in the third exon of the human BIC gene.22
MiR-155 plays a crucial role in the development, function and regulation of immune cells, including B-cells, T-cells and dendritic cells.23-27 Aberrant expression of miR-155 is involved in the pathogenesis
of several autoimmune diseases, e.g. rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus.27,28 The oncogenic potential of BIC/miR-155 is supported by its high expression in
many B cell lymphoma subtypes and by the development of cancer in transgenic mice.29-32 Interestingly,
miR-155 may also function as a tumor suppressor by regulating the expression of Activation-Induced Cytidine Deaminase (AID) and thereby limiting or even preventing the occurrence of Myc-Igh translocations in activated B cells.33 In line with this finding, miR-155 is expressed at very low levels
5
II. miRNA profiling studies in Hodgkin lymphoma
In Hodgkin lymphoma, both miRNA profiling and functional studies have been hampered due to the scarcity of the tumor cells in the affected tissue samples and the lack of appropriate animal models. Studies on HL pathogenesis frequently depend on the use of Hodgkin lymphoma cell lines or total tissue samples. The total number of Hodgkin lymphoma cell lines is limited, but includes the main cHL subtypes of both B and T cell origin and one NLP HL cell line. These cell lines are usually derived from end-stage and progressive or relapsed Hodgkin lymphomas and are thus not entirely representative of HRS and LP cells at the time of diagnosis. Currently published miRNA profiling studies have been performed using these HL cell lines and also on microdissected HRS cells and total tissue sections.34-38
a. HL cell lines
Landgraf et al sequenced 250 small RNA libraries including four cHL cell lines and various normal B cell subsets as well as other hematological cell lines.34 Three of the four cHL cell lines
that were EBV- clustered together and were found to most closely resemble the plasmacytoma cell lines. The fourth HL cell line was EBV+ and clustered together with other EBV immortalized lymphoma derived cell lines. A very high expression level was observed specifically for miR-16, miR-21, miR-29b, miR-142, miR-155 in the three EBV- cHL derived cell lines.
Lawrie et al also analyzed miRNA expression in four HL cell lines and compared the expression pattern profile of B-cells of different developmental stages.36 Two of the four HL cell lines (L428
and L1236) clustered closely together. These two cell lines also clustered closely together with a primary mediastinal B cell lymphoma cell line, a lymphoma subtype which was shown to have many similarities with HL in gene expression studies. KM-H2 clustered separately and the T cell derived L540 HL cell line clustered closely together with ALK+ anaplastic large cell lymphoma (ALCL). Three of the four cell lines were also included in the study by Landgraf et al and a direct comparison by Lawrie et al showed a comparable expression pattern.
Gibcus et al subsequently profiled three B cell derived cHL cell lines and one B cell derived NLP HL cell line.37 A high expression was observed for the oncogenic miR-17-92 cluster,
miR-16, miR-21, miR-24 and miR-155 in the three cHL cell lines. Several miRNAs were differentially expressed in cHL as compared to either EBV transformed lymphoblastoid cell lines, primary mediastinal B cell lymphoma and Burkitt lymphoma cell lines. The most pronounced differences were observed between cHL and BL cell lines with 16 differentially expressed miRNAs, including upregulation of miR-155 and miR-9 and downregulation of miR-150. Downregulation of miR-150 was observed also in comparison to LCL and PMBL cell lines. The overlap between the most abundantly expressed miRNAs in this study and the most abundantly expressed miRNAs in the small RNA sequencing libraries of Landgraf et al consisted of 18 miRNAs (listed in Table 1).
In addition to the cell lines reported in the study by Gibcus et al, we now also profiled five additional lymphoma cell lines and purified primary GC B cells (Figure 1). Unsupervised clustering revealed one cluster that contained the cHL cell lines with a confirmed B-cell origin based on immunoglobulin rearrangements and PMBL cell lines, a clustering pattern similar to that observed by Lawrie et al. A second cluster contained the nodular lymphocyte predominant (NLP) HL cell line DEV, normal GC B cells and part of the diffuse large B cell lymphoma cell lines. The other 3 cHL cell lines (L540 and HDLM2 of confirmed T-cell origin and SUPHD1 of uncertain origin) cluster separate from the other cHL cell lines and in part together with Burkitt lymphoma and diffuse large B cell lymphoma cell lines.
b. Microdissected HRS cells
There is one miRNA profiling study that used laser microdissection to isolate primary HRS cells from patient material. Thirty microdissected primary HRS cells per patient were pooled and RNA was isolated to generate miRNA signatures for nine cHL patients.38 In comparison
to CD77+ B cells 41 miRNAs were differentially expressed. Fifteen of these miRNAs were also differentially expressed in cHL cell lines in comparison to CD77+ B cells; 12 miRNAs were upregulated and three miRNAs were downregulated. The overlap with our analysis (as shown in Figure 1) comprises three miRNAs, i.e. miR-9, miR-16 and miR-18a. The relatively small overlap between both studies might be explained by different strategies applied to sort GC B cells. Six of the 12 upregulated differentially expressed miRNAs were also among the most abundantly expressed miRNAs in the cell lines, i.e. 9, 16, 20a, miR-21, miR-30b and miR-155 (Table 1). Consistent findings with respect to the most abundantly expressed miRNAs observed in the studies by Landgraf et al and Gibcus et al included miR-16, miR-20a, miR-21 and miR-155.
c. Total tissue samples
The second study using primary cases analyzed total tissue samples of 49 cHL patients and 10 reactive lymph nodes.35 A signature of 25 miRNAs could differentiate cHL from reactive lymph
node tissue, whereas 36 miRNAs were differentially expressed between nodular sclerosis and mixed cellularity subtypes. For four miRNAs, i.e. miR-21, miR-134, miR-138 and miR-155, in-situ hybridization confirmed expression in the HRS cells. 20 of the 25 differentially expressed miRNAs between cHL and lymph node were also highly expressed in cHL cell lines, suggesting a tumor cell specific expression pattern. Comparing the top-45 most abundantly expressed miRNAs in total tissue samples of this study to the most abundant miRNAs observed in primary HRS cells or cell lines in the previously discussed studies revealed only a marginal overlap (Table 1). This difference is most likely explained by the analysis of total tissue including only a minority of tumor cells and a vast majority of inflammatory cells. Navarro et al. also compared EBV+ and EBV- cHL tissue samples and found three miRNAs, i.e. miR-96, miR-128a, and miR-128b that were specifically downregulated in nodular sclerosis EBV+ cHL as compared to nodular
5
sclerosis EBV- cHL.35 These differences might reflect putative differences in microenvironment
or indirect effects caused by EBV proteins or miRNAs. No specific information was given on the expression of EBV derived miRNAs.
IV. Functional miRNA studies in HL
A substantial number (>25) of miR-155 target genes have been identified in different normal and malignant cell types in the past few years. Some of these proven targets, i.e. AID, FOXO3a, IL13Ra1, PU.1, SHIP1 and SOCS1, have been shown to be involved in the pathogenesis of HL. 39-44 However, to date there are no studies that have addressed the functional consequences of
the high miR-155 levels in HL on these proven target genes.
Gibcus et al. tested the 3’UTR sequence of 11 previously experimentally validated target genes by luciferase reporter assays in three HL cell lines and observed a miR-155 dependent targeting for 6 genes, i.e. ZIC3, AGTR1, ZNF537, KGF, MAF and IkBkE, in one or more of the cell lines.37
No further validation at the protein level of these target genes has been done. A recent study by Dagan et al. showed that HGAL is also a direct target of miR-155 in diffuse large B cell lymphoma.45 HGAL causes decreased lymphocyte and lymphoma cell motility by activating the
RhoA signaling cascade. In HL, miR-155 levels are high and HGAL levels are typically low, which indicates that HGAL might be a pathogenetically relevant target gene of miR-155 in HL.46
In a ribonucleoprotein immunoprecipitation Chip (RIP-Chip) experiment using antibodies against Ago2, a comprehensive overview of miRNA targets was generated for two Hodgkin lymphoma (HL) cell lines. A significant overrepresentation of genes involved in proliferation, apoptosis and the p53 pathway were identified in the Ago2 IP-fraction, indicating that these processes are regulated by miRNAs in HL.47 A high proportion of the miRNA target genes
were regulated by the miR-17 seed family. Validation of 11 of the miR-17 identified targets by luciferase reporter assay indicated a miR-17 dependent regulation for nine genes, e.g. ZNFX1, CCL1 and GPR137B. In a follow up study, Gibcus et al showed that CDKN1A encoding the p21 protein - one of the target genes identified by RIP-ChIP analysis - was a valid miR-17 seed family target gene in HL cell lines using luciferase reporter assay and Western blotting.48 Inhibition of
the miR-17 seed family using antisense oligonucleotides resulted in increased p21 levels and a block in the G1-S cell cycle transition.
Figure 1. Heatmaps of microRNA profiling study in HL, non-Hodgkin lymphoma derived cell lines and
normal GC B cells. 8 HL and 11 non-Hodgkin lymphoma cell lines were analyzed on a Agilent miRNA microarray platform (version V1). Germinal center B cells (GCB) purified from three individuals (CD19+, IgD-, CD38+) were analyzed as their normal counterparts. A. Heatmap of unsupervised clustering of the 151 miRNAs flagged present in at least all samples of one sample subset. B. Enlargement of the clustering pedigree from part A. The B cell classical (c)HL cell lines cluster together with the PMBL cell lines, whereas the two T cell cHL cell lines (HDLM2 and L540) and one cHL of uncertain origin (SUPHD1) cluster separately. The NLPHL cell line DEV clustered together with the normal GC B cells. C. Heatmap of significantly differentially expressed miRNAs between B cell cHL and GC B cells. D. Heatmap of significantly differentially expressed miRNAs between B cell cHL and BL. E. Heatmap of significantly differentially expressed miRNAs between B cell cHL and PMBL. F. Heatmap of significantly differentially expressed miRNAs between B cell cHL and DLBCL. (unpaired T-test, miRNAs with p-value of <0.01 are shown).
5
Table 1. Overview of the most abundantly expressed miRNAs observed in four HL profiling
studies miRNA cHL cell lines (top-30) Landgraf cHL cell lines (top-27) Gibcus HRS cells and cHL cell lines (top-6) Vlierberghe Total tissue samples (top-45) Navarro let-7a + + - -let-7b + - - -let-7f + + - -let-7g - + - -let-7i + - - + miR-103 - + - -miR-106a - + - + miR-106b + + - -miR-140 + - - -miR-142-3p + + - -miR-142-5p + + - + miR-155 + + + -miR-15a + + - -miR-15b - + - -miR-16 + + + -miR-17 + + - -miR-186 + - - -miR-18a + - - -miR-191 + + - -miR-195 + - - -miR-19a + + - -miR-19b + + - -miR-20a + + + -miR-20b - + - -miR-21 + + + + miR-24 + - - -miR-25 - + - -miR-27a + - - + miR-27b + - - -miR-29a + + - -miR-29b + + -
-Table 1. Continued miRNA cHL cell lines (top-30) Landgraf cHL cell lines (top-27) Gibcus HRS cells and cHL cell lines (top-6) Vlierberghe Total tissue samples (top-45) Navarro miR-29c - + - -miR-30b - + + -miR-30d + - - -miR-30e + - - -miR-425 + - - -miR-565 - + - -miR-9 + - + + miR-92a + + - -miR-93 + + - -- - - 39 non-consistent miRNAs +, present in top-list; -, not present in top-list
A second functional study in HL focused on miR-9 and let-7a targeting of PRDM1/Blimp-1, a master regulator in terminal B cell differentiation.49 High levels of miR-9 and let-7a correlated
with low levels of PRDM1 in HL cell lines. The majority of HRS cells in primary HL cases also showed weak or no PRDM1/Blimp-1 expression. Inhibition of miR-9 or let-7a in HL cell lines using antisense oligonucleotides resulted in reduced PRDM1/Blimp-1 levels. It can be speculated that high miR-9 and let-7a levels prevent plasma cell differentiation of HRS precursor cells and thereby contribute to the pathogenesis and phenotype of HRS cells. In a very recent paper, Leucci et al also studied the role of miR-9 in Hodgkin lymphoma.50 Inhibition of miR-9
resulted in increased mRNA levels of DICER1 and HuR. Subsequent luciferase and Western blot analysis confirmed targeting of these two genes by miR-9. Besides induction of these two genes, the authors also observed significant differences of genes that have a consensus HuR binding motif. HuR can bind to AU rich transcripts and prevent degradation via the AU-mediated decay pathway. These binding motifs are found in the transcripts of many cytokines and chemokines and the authors showed a HuR dependent effect of miR-9 inhibition on the chemo-attracting potential of HL cell line culture medium. Moreover, effects of miR-9 inhibition were observed on TNFa, CCL5, IL-5 and IL-6 expression. In a xenograft mouse model, reduced tumor outgrowth was observed upon subcutaneous delivery of miR-9 inhibitors.<sup>51</sup> It is unclear whether these effects are caused by targeting of PRDM1, HuR or one or more of the other miR-9 target genes.
5
Based on the prognostic value of miR-135a in cHL (see below), Navarro et al studied miR-135a target genes in HL.52 Overexpression of miR-135a in HL cell lines resulted in increased caspase
levels and decreased growth of the cells. The cytoplasmic tyrosine kinase JAK2 contained a miR-135a binding site in the 3’-UTR and a direct regulation was shown using a luciferase reporter assay and by Western blot. Overexpression of miR-135a induced downregulation of the JAK2 protein levels and as a consequence Bcl-xL was also downregulated in HL. This suggests a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis.52
In a recent study Xie et al. showed a consistent downregulation of FOXO1 in both cHL and NLPHL.53 The FOXO1 locus at 13q14 was frequently deleted in HL cell lines and HRS cells purified
from primary HL tissue samples. A second factor that contributed to the downregulation of FOXO1 was the constitutively activated PI3K/AKT and ERK pathways. A third factor associated with the low FOXO1 levels was the high expression of miR-96, miR-182 and miR-183, three miRNAs previously shown to target FOXO1.54 A marked induction of FOXO1 protein levels
was shown upon inhibition of these miRNAs in HL cell lines, confirming an effective targeting. Reintroduction of FOXO1 induced a growth arrest and apoptosis in all five cHL cell lines studied and indicated a functional role of FOXO1 downregulation in the pathogenesis of HL.
V. Clinical value of miRNAs in HL
To date, potential clinical applications of miRNAs include their use as prognostic markers, biomarkers or as therapeutic targets. Many studies already report on the value of miRNAs as prognostic, tissue or serum biomarkers in different malignancies.55 There is currently only one
study that analyzed the prognostic value of miRNAs for HL patients.52 In this study a group
of 89 cHL patients was analyzed; 76 patients had a complete remission, 5 a partial remission, and 8 patients were chemoresistant. Of 25 miRNAs tested a significant difference in survival was observed only for miR-135a by comparing patients with a high level (n=42) of this miRNA to patients with a low level (n=22). Patients with a low miR-135a level had a shorter disease free survival (P = .02) and showed a higher frequency of relapse (P = .04), consistent with its proposed biological role in inhibiting apoptosis by modulating JAK2.52 In multivariate analysis
miR-135a remained a prognostic factor for disease free survival. Given the relatively small patient cohort, it is essential to validate these findings in other larger cohorts to establish the true prognostic value of miR-135a.
VI. Concluding remarks and future perspectives
It is obvious that miRNAs are involved in the pathogenesis of HL and can have an effect on different cellular pathways (Figure 2). Only a few studies show functional effects upon miRNA induction or inhibition that directly affect cell growth and apoptosis. These miRNAs, i.e. miR-9, the miR-17 seed family and miR-135a, might thus be considered as potential therapeutic targets for the treatment of HL. For the highly expressed miR-155 the direct pathobiological
consequences remain unknown and additional studies are required. In many malignancies the use of circulating miRNAs as possible prognostic or disease markers has been studied, but the value of circulating miRNAs in HL is still unknown. Currently, the potential for clinical applications of miRNA in HL is still limited and more studies are needed to fully explore the strength and breadth of opportunities.
miR-155 ZIC3 AGTR1 ZNF537 KGF MAF IκBκε HRS cell pri-miRNA pre-miRNA DGCR8 DROSHA BIC
miR-9 DICER HUR cytokine production / proliferation
miR-17 seed-family CDKN1A ZNFX1 CCL1 GPR137B let-7a PRDM1 / BLIMP-1
miR-135a
JAK2 Inhibition of apoptosis through Bcl-xL miR-96 miR-182
miR-183
FOXO1 Growth arrest / apoptosis Exportin-5 DI CE R RISC plasma cell differentiation block G1-S cell cycle checkpoint Prognostic value
Figure 2. Schematic presentation of the currently proven miRNA targets in HL and their pathogenetic
5
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