miR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma

doi:10.21203/rs.3.rs-2496311/v1

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miR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma

https://doi.org/10.21203/rs.3.rs-2496311/v1

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B-cell receptor (BCR) signaling is critically activated and targetable for mantle cell lymphoma (MCL); however, the underlying mechanism of the activated BCR signaling pathway is not clear. The pathogenic basis of miR-17-92 cluster remains unclear although the oncogenic microRNA (miRNA) miR-17-92 cluster is highly expressed in patients with MCL. This study revealed that miR-17-92 cluster overexpression is partly dependent on SOX11 expression and chromatin acetylation of MIR17HG enhancer regions. Moreover, miR-17-92 cluster regulates not only cell proliferation but BCR signaling activation in MCL cell lines. Pulldown-seq, where mRNA was captured using biotinylated miRNA transfection, was performed and analyzed with next-generation sequencing. Additionally, novel miRNA targets, including tumor suppressors such as BTG2, were identified to comprehensively define miR-17-92 cluster targets. Notably, gene expression profile data of patients with MCL revealed that BTG2 expression was negatively associated with those of BCR signature genes. Moreover, BTG2 silencing in MCL cell lines significantly induced BCR signaling overactivation. Our results suggest an oncogenic role of miR-17-92 cluster-activating BCR signaling throughout BTG2 deregulation in MCL.

Biological sciences/Cancer/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma

Biological sciences/Cancer/Oncogenes

mantle cell lymphoma

miR-17-92 cluster

B-cell receptor signaling

BTG2

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma (BCL) that accounts for 3–6% of all malignant lymphomas [1]. The advent of rituximab-containing immunochemotherapy and molecular-targeted therapeutics has improved the MCL outcome. Notably, targeting therapy for Bruton’s kinase (BTK), which is a component of the B-cell receptor (BCR) signaling pathway, such as ibrutinib, acalabrutinib, and zanubrutinib, is highly effective for MCL. However, MCL remained incurable despite the recent development of novel therapy. Moreover, the prognosis is generally very poor when MCL progress to be refractory to target therapy, such as BTK inhibitors [2, 3].

The mechanisms of BCR activation in MCLs are not sufficiently understood although BCR signal activation is important for MCL pathogenesis [3–6]. Genomic abnormalities in various genes that compose BCR signaling pathway, such as TNFAIP3, CARD11, and MYD88, are considered drivers of BCR signal activation in other types of BCL, such as diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) [7–11]. In contrast, these genetic abnormalities are uncommon in MCL. Some MCLs show reactivity to autoantigens, suggesting chronic active BCR signaling activation [12, 13]. SOX11 overexpression and MCL tumor microenvironment have induced BCR activation [12]. However, the detailed mechanisms of BCR activation remained unclear. Hence, knowledge of the pathogenic basis of BCR signal activation is urgently needed to overcome the resistance to BCR signal-targeted therapies.

MicroRNAs (miRNAs) are small 20–24 nucleotide non-coding RNAs that induce mRNA degradation and suppress translation [14]. miR-17-92 cluster consists of six miRNAs, including miR-17, 18a, 19a, 20a, 19b-1, and 92a-1, located on chromosome13q31.3, with two paralogs, i.e., miR-106a-363 and miR-106b-25 [15, 16]. These miRNAs play oncogenic roles in various cancers [17]. Additionally, miR-17-92 cluster is constitutively overexpressed in MCLs, and miR-18b overexpression is associated with poor prognosis [18–21]. However, the function and target genes of miR-17-92 cluster in MCL are not well known.

This study explored the fundamental roles of the miR-17-92 cluster through comprehensive analysis to identify the targets of these miRNAs in vitro and revealed that BTG2, which is a novel target of miR-17-92 cluster, regulates the proliferation and BCR signaling activation in MCL.

Cell lines and reagents

This study used four human MCL cell lines, including Jeko-1, JVM2, and Z138 obtained from American Type Culture Collection and KPUM-YY1 established from a patient with MCL in our laboratory [22]. Cells were maintained in RPMI-1640 (Wako, Osaka, Japan) containing 10% fetal calf serum (Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamate, and penicillin/streptomycin at 37℃ in humidified 95% air and 5% CO₂. Normal B lymphocytes were collected from the blood of two healthy donors to analyze the expression level of miR-17-92 cluster. CD19-positive B lymphocytes were isolated using Lymphocyte Separation Solution (Nacalai Tesque, Inc., Kyoto, Japan) and MACSprep™ Chimerism CD19 MicroBeads, human (Miltenyi Biotec, Bergisch Gladbach).

Transfection Of Mirna Mimics And Inhibitors

MCL cells were transfected with 1 µM miRCURY LNA™ Premium miRNA Mimic, miRCURY LNA™ microRNA Mimic Negative Control (Qiagen, Hilden, Germany), 6 µM mirVana™ miRNA inhibitor, or mirVana™ miRNA inhibitor negative control #1 (Life Technologies) using Hemagglutinating Virus of Japan (HVJ)-envelope vector (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan), following the manufacturer’s protocol.

Pulldown-seq

The protocol was modified by previous reports [24–26]. The biotinylated miRNA mimic or negative control was introduced into Z138 cells, and target mRNA was captured (Fig. 3A). The complex of captured target mRNA and miRNA mimic was pulldown with magnetic beads-coated streptavidin. The extracted RNA was sequenced and analyzed by next-generation sequencing (NGS) at the NGS Core Facility of the Kyoto Prefectural University of Medicine. Details are described in Supplementary Methods.

Ca Flux Assay

The strength of the BCR signaling was quantified by measuring intracellular Ca²⁺ concentration. Cells were incubated with 3 µM Fluo-4AM (Dojindo Molecular Technologies, Kumamoto, Japan) in Hanks’ balanced salt solution (HBSS) with CaCl₂ at 37℃ for 45 min. Cells were subjected to flow cytometric analysis using a FACS Celesta (BD Biosciences, San Jose, CA, USA) after washing the cells with HBSS without CaCl₂. The measuring tube was removed 30 s after the start of the measurement, 10 µg/mL of anti-IgM (Jackson ImmunoReserch Laboratories Inc., PA, USA) was administered at 35 s, and measurements were restarted from 40 to 340 s. The FlowJo™ software (BD Biosciences) was used to analyze data. The signal intensity was normalized with the means of those between 0 and 30 s, and the area under the curve (AUC) between 42.5 and 242.5 s was calculated. These experiments were performed in triplicate.

Fluorescence In Situ Hybridization (Fish)

A dual-color interphase FISH study was performed to detect 13q31.3 amplification, which contains the MIR17HG locus. FISH was conducted as previously described [27]. Bacterial artificial chromosome clones (Advanced Geno Techs Co., Ibaraki, Japan) of RP11-97P7 and RP11-936K15 were used to generate probes for MIR17HG (13q31.3) and centromeric region (13q12.11), respectively.

Gene Silencing Using Lentiviral Infection

SOX11 and BTG2 silencing were accomplished by stable expression of inhibitory short hairpin RNAs (shRNAs). The target sequences for human SOX11 and BTG2 were 5'-GCC TCT ACT ACA GCT TCA AGA-3' (shSOX11#1), 5'- GCT GTT ATC TTA GTT TAA AGA-3' (shSOX11#2), 5'-GCT GCA TTC GCA TCA ACC ACA-3' (shBTG2#1), and 5'-GCA TTC GCA TCA ACC ACA AGA-3' (shBTG2#2). The sequences encoding shRNAs against SOX11 and BTG2 were cloned into lentivirus vector pLKO.1 puro (Addgene, Watertown, MA, USA). The empty vector was used as negative control (mock). Lentivirus was produced by transient co-transfection of HEK293T cells with a lentivirus vector, packaging plasmid pMDLg/pRRE, pRSV-Rev, and pMD2.G using PEI MAX (Polysciences, Warrington, PA, USA). Viral supernatant was collected at 48 and 72 h after transfection and was concentrated by ultracentrifugation. Infected MCL cells were selected with appropriate concentrations of puromycin from 48 h after infection and maintained under puromycin selection for 2 weeks in BTG2 silencing.

Microarray Data Analysis

Publicly available and deposited gene expression data in National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO; accession number GSE93291) were analyzed to investigate the relationship between BTG2 gene expression level and MCL clinical characteristics. The expression data were normalized with the robust multichip array method. One with the higher mean signal value from multiple probes was chosen for each gene.

The overall survival was compared within two subgroups defined by the BTG2 signal with the cut-off of the median, using the log-rank method. BCR signature gene expression was evaluated using the gene set from Herishanu, et al [28].

Statistical analysis

Statistical analyses were performed with R (version 4.0.5). The student’s t-test was used to compare continuous variables between groups. A p-value of < 0.05 was considered significant.

Other methods are described in Supplementary Methods.

Mechanism of miR-17-92 cluster overexpression in MCL

First, each miR-17-92 cluster miRNA expression was confirmed to be significantly higher in the cell line than in normal B lymphocytes using qRT-PCR (Supplementary Fig. 1). The mechanism of miR-17-92 cluster overexpression in MCL is not well understood although amplification or gain of 13q31-32, where MIR17HG is located, are found in 10–20% of MCLs [29]. We examined FISH analysis and revealed that none of the cell lines evaluated in this study harbored copy number alterations in 13q31 (Supplementary Fig. 2). Next, we hypothesized that SOX11 regulates miR-17-92 transcription because previous data of ChIP-Seq of SOX11 in MCL cell lines showed that SOX11 binds upstream of the MIR17HG locus [30]. Silencing SOX11 repressed not only cell proliferation but the expression of each miR-17-92 in two of three SOX11-positive MCL cell lines, i.e., Jeko-1 and Z138 (Fig. 1A-C). Additionally, we analyzed using BRD4 ChIP-seq data of SOX11-negative JVM2, which we had previously performed (Fig. 1D) [31]. High BRD4 binding suggesting histone acetylation was found in the MIR17HG enhancer region in JVM2 cells although H3K27Ac ChIP-seq publicly available data of naive B cells and germinal center B cells in healthy human tonsil revealed no enrichment of acetylated histone marks upstream of MIR17HG.

Functional Role Of Mir-17-92 Cluster In Mcl Pathogenesis

We performed a proliferation assay in MCL cell lines transfected with miRNA inhibitors, which are chemically modified, single-stranded oligonucleotides designed to specifically bind to endogenous miRNAs, to confirm the oncogenic role of these miRNAs in MCL. The cell proliferation of inhibitor-transfected cell lines was modestly repressed compared to the negative control group in all MCL cell lines examined (Supplementary Fig. 3). The relative inhibition of proliferation by individual miRNA inhibitors varied among cell lines, and the results were not consistent. The underlying mechanism affecting cell proliferation is not well revealed despite miR-17-92 cluster being known as oncogenic miRNA. Then, we hypothesized that miR-17-92 cluster might be associated with BCR signal pathway upregulation because both miR-17-92 cluster and the BCR signal are aberrantly activated in MCL cells compared to the normal counterpart of naive B cells. We evaluated intracellular Ca²⁺ influx from ER stores in MCL cell lines crosslinked and stimulated with anti-IgM BCR to assess the impact of miR-17-92 cluster on the BCR signal (Fig. 2A). The calcium response upon BCR stimulation was significantly reduced in cells transfected with all miRNA inhibitors except for miR-17, which revealed a tendency of inducible effect on the BCR signal although not statistically significant (Fig. 2B). These results suggest that miR-17-92 cluster leads to cell growth promotion partly depending on BCR signal activation.

Investigation Of Targeted Genes Of Mir-17-92 Cluster

The landscape of miR-17-92 cluster targets, especially in MCL, is not described although a part of the targets of the miR-17-92 cluster consists of several genes, such as FGFR3B and CD22 affecting BCR signal in other lymphomas such as DLBCL. Moreover, miRNA targets are different among tissues and cancer types. Therefore, this study aimed to identify the target genes of each matured miRNA of the miR-17-92 cluster in MCL cells but not by in silico simulation. We performed pulldown-seq, where we captured mRNA using biotinylated miRNAs, and analyzed them with NGS to comprehensively identify miR-17-92 cluster-targeted genes (Fig. 3A). We used the cell line, Z138, which had the lowest expression of miR-17-92 cluster among the cell lines used in this study, to minimize the impact of overexpression on endogenous miRNAs (Supplementary Fig. 1). The level of each miRNA was checked by RT-qPCR against negative control to determine whether the pulldown was successful (Supplementary Fig. 4). Quantitative analysis of captured mRNA level comparing those of each miRNA mimic and negative control identified candidate targets, some of which are known as recurrently mutated genes in MCL, BCR signal-related genes, and tumor suppressors as follow: BTG2 (miR-19b), CDKN2A (miR-17), FCGR2A (miR-17), SYNE1 (miR-20a), TET2 (miR-18, miR-19b, and miR-20a), TNFRSF10A (miR-20a), and TRAF3 (miR-17) (Fig. 3B; Supplementary Table 1).

Functional Analysis Of Btg2 In Mcl

We adopted publicly available gene expression profile data derived from patients with MCL who received R-CHOP therapy (GSE93291) to investigate the clinical significance of those candidate target genes in patients with MCL [32]. Data of these candidate genes revealed that the expression of BTG2 was negatively correlated with those of BCR signature genes (P = 1.15e-04, R = 0.342) (Fig. 4A). Moreover, low BTG2 expression was significantly associated with poor overall survival (P = 0.006) (Fig. 4B). Therefore, we inferred the important role of BTG2 in BCR signaling and MCL pathogenesis.

We evaluated the BTG2 expression at the protein level to validate the data of pulldown-seq showing that BTG2 was a candidate for the target of miR-17-92 cluster, and confirmed BTG2 upregulation after the introduction of miR-17-92 cluster inhibitors in all four MCL cell lines examined (Fig. 5). Increased protein level expression of BTG2 was observed in miR-17 and miR-18a, which could not be identified by pulldown-seq, in common with the four cell lines. We knocked down BTG2 by RNA interference (RNAi) in four MCL line cells to explore the functional role of BTG2 (Fig. 6A-B; Supplementary Fig. 5A-B). Under normal culture conditions, no difference was found between mock and knockdown cells in the three cell groups examined (Supplementary Fig. 6A). We tried the proliferation assay under starvation condition, because BTG2 is a known stress-induced gene. Cell proliferation relatively increased in the knockdown group compared to the mock group in the three cell groups examined, excluding KPUM-YY1, which could not tolerate reduced FBS medium, when serum was reduced from 10–2% in the medium (Supplementary Fig. 6B). Moreover, Ca²⁺ flux assay revealed that BTG2 knockdown enhanced BCR signaling (Fig. 6C; Supplementary Fig. 5C). The molecules that constitute the BCR signaling involved upon BTG2 knockdown were examined in WB. Additionally, the involvement of the Syk, BTK, and NF-κB pathways was considered. Conversely, Lyn showed little change between mock and knockdown groups, suggesting that it was unregulated by BTG2 (Fig. 6B).

This study revealed that miR-17-92 cluster, which is known as overexpressed oncogenic miRNA in MCL, regulates BCR signaling. Moreover, we identified BTG2 as a novel target of miR-17-92 cluster, and importantly, the BTG2 downregulation led to BCR signal activation.

miRNAs are small non-coding RNAs that silence endogenous target genes throughout mRNA degradation and translation inhibition. Targeting of miRNAs generally depends on the interaction between seed sequences of miRNAs consisting of 7–8 nucleotides and complementary sequences mainly in 3'UTR of mRNAs. However, the recent development of sequencing approaches, such as argonaute cross-linking immunoprecipitation (AGO-CLIP), demonstrated that miRNA targets are determined by not only the canonical interaction of 3'UTR of mRNAs but 5'UTR and coding region [33]. Therefore, the classical approach to identifying the targets of miRNAs using mRNA expression analysis and in silico predicting tools using miRNA sequences might not fully capture the targets of each miRNA. Moreover, target mRNAs of each miRNA potentially vary among the cancer types because of the different endogenous expression levels of mRNAs and miRNAs, which constitute complex regulatory networks with each other. Therefore, our pulldown-seq approach is suitable for comprehensive analysis of miR-17-92 cluster in MCL. BTG2 was identified as a potential target for miR-17, miR-20a, and miR-92a in the TargetScan (https://www.targetscan.org/vert_80/) and miRDB (https://mirdb.org/), whereas no study revealed that BTG2 is a direct target of these miRNAs to date. Our study revealed that other miRNAs constituting miR-17-92 cluster also targeted BTG2.

BCL subtypes are dependent on different pathogenic BCR signal modes. The tonic BCR signal in GCB-DLBCL and FL is activated to engage the PI3K pathway [13, 34]. Chronic active BCR signal, which engages BCR-dependent NF-κB and PI3K signal, is activated under the genetic alterations, such as CD79A/CD79B, CARD11, MYD88 mutations, and loss of TNFAIP3, in ABC-DLBCL [7–11]. Chronic active BCR signal in MCL is generally considered active. IGH-V restriction in MCL suggests self-antigen-driven BCR signal activation and a part of patients with MCL harbors LRPAP1 self-antibody [35]. SOX11 overexpression and CXCR4-dependent microenvironment interaction are considered important for BCR signal activation [36]. However, the background of BCR signal activation is not sufficiently resolved. miR-17-92 cluster overexpression has been previously reported to activate the PI3K/AKT/mTOR pathway by targeting the protein phosphatase PHLPP2, PTEN, and BIM in MCL [37, 38]. In DLBCL, miR-17-92 cluster in the presence of Myc enhances BCR signaling activity via ITIM-containing proteins such as CD22 and FCGR2B [19]. Our study could not capture all of miR-17-92 cluster targets associated with the BCR pathway in the pulldown-seq analysis, partly because of the sensitivity of this method and the difference of dependency of these molecules for the BCR signal in Z138 cells which we used.

BTG2 is one of the antiproliferative (APRO) gene family (TOB1-2, BTG1-4), which is down-regulated in many cancers as a tumor suppressor gene and is involved in cell differentiation, proliferation, DNA repair, and apoptosis [39, 40]. The underlying mechanism of BTG2 downregulation is not fully described, and our findings illustrate one of the causes for tumor-specific decreased BTG2 expression. Regarding the function of BTG2 in blood cells, BTG2/protein arginine methyltransferase-1 (PRMT1) complex is generally generated in B cells, and CDK4 methylation and CCND3 inhibition lead to cell proliferation and differentiation inhibition through cell cycle arrest [41, 42], and it is involved in mRNA stability in T cells [43].

No reports examined the function of BTG2 in malignant lymphoma pathogenesis although BTG2 mutations are frequently found in several lymphomas, such as DLBCL and FL [44], and this mutation was reported as a poor prognostic factor in primary testicular diffuse large B-cell lymphomas [45]. Regarding the mechanism by which BTG2 regulates BCR signaling, PRMT1, which interacts with BTG2 and catalyzes asymmetric dimethylation of arginine-residues localized within glycine arginine-rich regions, has been previously reported to suppress BCR signaling through CD79A R198 methylation just below the ITAM region [42, 46, 47]. BTG2 inhibition and PRMT1 interaction in the MCL results in CD79A methylation inhibition and BCR signaling activation.

The mechanism of miR-17-92 cluster overexpression in MCL is not well understood although 13q31-32 amplification or gain, where MIR17HG is located, is found in 10–20% of MCLs. We found that SOX11 overexpression and enhancer-regulated mechanism induced overexpression of miR-17-92 in a part of MCL cell lines. The detailed mechanism of SOX11-dependent BCR activation is unknown although previous studies demonstrated that SOX11-overexpressed B lymphocytes develop MCL-like lymphoma under BCR activation [48]. Our result revealing that SOX11 regulates miR-17-92 suggests a part of the cause of SOX11-dependent BCR activation. Further investigation, especially using patient-derived samples, is needed.

The role of BTK Inhibitors, such as ibrutinib in MCL treatment, is currently significant, and elucidating the mechanism of BCR signaling activation in MCL is an important issue in overcoming the clinical unmet need for targeted therapy resistance in this pathway. This study, which revealed that miR-17-92 cluster activates BCR signaling by regulating BTG2 in MCL, has the potential to contribute to future investigations of therapeutic response and improvement of therapeutic outcomes in MCL. Furthermore, the comprehensive analysis of the target molecules of miR-17-92 cluster are expected to elucidate the pathogenesis of not only MCL but also many other malignant tumors.

Acknowledgments

The authors thank N. Sakamoto-Inada for her excellent technical assistance. The study was supported in part by grants from Nippon Shinyaku Research Grant and JSPS KAKENHI (19K17867) (TT).

Author Contributions

YK-K, TT, MN, YT, HN, KM, YK-T, RI, TF, YM-K, SM, and YS performed experiments. TT, MN, and YT analyzed NGS data. YK-K, TT, MN, YT, and JK drafted the manuscript. TT and JK designed the study. MT, KT, and JK supervised the research.

Competing Interests

JK received research funding from Kyowa Kirin, Chugai Pharmaceutical, Daiichi Sankyo Pharmaceutical, Ono Pharmaceutical, Eisai, Taiho Pharmaceutical, Sumitomo Pharma, Shionogi Pharmaceutical, and Bristol Myers Squibb, has received honoraria from Ono Pharmaceutical, Sanofi, Bristol Myers Squibb, and Janssen Pharmaceutical, and is a consultant for Janssen Pharmaceutical, Bristol Myers Squibb, Asahikasei Pharma, and Pfizer.

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miR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma

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Abstract

Figures

Introduction

Materials And Methods

Cell lines and reagents

Transfection Of Mirna Mimics And Inhibitors

Pulldown-seq

Ca Flux Assay

Fluorescence In Situ Hybridization (Fish)

Gene Silencing Using Lentiviral Infection

Microarray Data Analysis

Statistical analysis

Results

Mechanism of miR-17-92 cluster overexpression in MCL

Functional Role Of Mir-17-92 Cluster In Mcl Pathogenesis

Investigation Of Targeted Genes Of Mir-17-92 Cluster

Functional Analysis Of Btg2 In Mcl

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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