Myeloma Cells Are Activated in Bone Marrow Microenvironment by the CD180/MD-1 Complex Which Senses Lipopolysaccharide

Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

KIKUCHI et al.

Myeloma Cells are Activated in Bone Marrow Microenvironment by the CD180/MD-1 Complex which Senses Lipopolysaccharide

Jiro Kikuchi1, Yoshiaki Kuroda1, Daisuke Koyama1, Naoki Osada1, Tohru Izumi2, Hiroshi Yasui3, Takakazu Kawase4, Tatsuo Ichinohe4 and Yusuke Furukawa1*

1Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan; 2Division of Hematology, Tochigi Cancer Center, Utsunomiya, Tochigi 320-0834, Japan, 3The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan, 4Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8583, Japan.

Running title: CD180 mediates LPS-induced growth of myeloma cells

Corresponding author: Yusuke Furukawa, Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel.: +81-285-58-7399; Fax: +81-285-44-7501; E-mail: [email protected]

Conflict of interest: The authors have no potential conflicts of interest

Notes about the manuscript: Word count 5905 excluding Materials and Methods, References and Author Contribution; Total numbers of figures 6

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Abstract

Multiple myeloma (MM) cells acquire dormancy and drug resistance via interaction with bone marrow stroma cells (BMSC) in a hypoxic microenvironment. Elucidating the mechanisms underlying the regrowth of dormant clones may contribute to further improvement of the prognosis of MM patients. In this study, we find that the CD180/MD-1 complex, a non-canonical LPS receptor, is expressed on MM cells but not on normal counterparts, and its abundance is markedly upregulated under adherent and hypoxic conditions. Bacterial LPS and anti-CD180 antibody, but not other TLR ligands, enhanced the growth of MM cells via activation of MAP kinases ERK and JNK in positive correlation with expression levels of CD180.

Administration of LPS significantly increased the number of CD180/CD138 double-positive cells in a murine xenograft model when MM cells were inoculated with direct attachment to BMSC.

Knockdown of CD180 canceled the LPS response in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the CD180 gene. Both cell adhesion and hypoxia activated transcription of the CD180 gene by increasing Ikaros expression and its binding to the promoter region. Pharmacological targeting of Ikaros by the immunomodulatry drug lenalidomide ameliorated the response of MM cells to LPS in a

CD180-dependent manner in vitro and in vivo. Thus, the CD180/MD-1 pathway may represent a novel mechanism of growth regulation of MM cells in a BM milieu and may be a therapeutic target of preventing the regrowth of dormant MM cells.

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Introduction

Multiple myeloma (MM) is characterized by deregulated growth of plasma cells, terminally differentiated B-lymphocytes, in the bone marrow (BM). Initial treatments with proteasome inhibitors and/or immunomodulatory drugs (IMiDs) significantly increased the remission rate of

MM; however, MM is still one of the most intractable malignancies due to a high incidence of relapse (1, 2). It is widely accepted that relapse stems from dormant and highly drug-resistant clones in the MM compartment. Drug resistant clones are kept dormant via interaction with bone marrow stroma cells (BMSCs) in a hypoxic microenvironment (3-6). Elucidation of the mechanisms underlying the regrowth of dormant clones may prolong remission and ultimately improve the survival of MM patients.

Recently, we have identified a novel epigenetic mechanism of drug resistance in which

BMSCs reduce the abundance of trimethylated histone H3 at lysine-27 (H3K27me3), a hallmark of condensed chromatin at silent loci, via EZH2 inactivation to derepress the transcription of several anti-apoptotic genes in MM cells (5, 6). The derepressed gene products include CD180, a homologue of Toll-like receptor 4 (TLR4), originally identified as 105 kDa protein (RP105) that imparts the resistance to radiation and corticosteroids in B-lymphocytes (7, 8). CD180 was later demonstrated to mediate the response to bacterial lipopolysaccharide (LPS), similar to TLR4 (9).

Toll-like receptors play a pivotal role in sensing and initiating innate immunity (10). The

TLR family is composed of 10 TLRs and CD180/RP105, each of which recognizes a specific pathogen-associated molecular pattern, leading to the activation of unique signaling pathways in immune cells. Some of them are also involved in oncogenesis and tumor growth (11). For example, MM cells express a broad range of TLRs and respond to specific ligands for proliferation and survival (12-15). Among the 10 TLRs, TLR4 is most prevalently expressed in MM cells.

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A study of a large panel of MM patients identified TLR4 overexpression in approximately 6% and its association with a poor treatment outcome (16). However, little is known about the role of

CD180/RP105 in MM biology.

In the present study, we found that CD180 is robustly expressed by MM cells but not normal plasma cells in the BM microenvironment in an Ikaros-dependent manner and senses bacterial

LPS to trigger the growth of dormant MM cells. Furthermore, the immunomodulatory drug lenalidomide could prevent the LPS-triggered activation of dormant clones by targeting CD180, providing a possible rationale for continuous and maintenance therapies of MM with this agent.

Materials and Methods

Reagents

The reagents used in this study and their sources are LPS (Wako Biochemicals, Osaka, Japan),

Pam3Csk (Novus, Littleton, CO), flagellin (Enzo Life Science, Am Arbor, MI), poke weed mitogen (Sigma-Aldrich, St. Louis, MO), lenalidomide, pomalidomide (Selleck Chemicals,

Houston, TX) and SN-50 peptide (Santa Cruz Biotechnology, Santa Cruz, CA) (17). Anti-CD180 antibodies with a stimulatory function (MHR73-11) and without a stimulatory function (N2C1) were obtained from BioLegend, San Diego, CA and GeneTex, Irvine, CA, respectively (18, 19).

LPS, Pam3Csk, flagellin, poke weed mitogen, and SN-50 were dissolved in 0.9% NaCl.

Lenalidomide and pomalidomide were dissolved in dimethyl sulfoxide at appropriate concentrations and used at a final dilution of 1/1000. The final concentration of dimethyl sulfoxide in the culture medium was <0.1%, a concentration that did not affect drug effects or cell growth per se.

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Cells and cell culture

We used 7 bona fide human MM cell lines, KMS12-BM, RPMI8226, KMS-21, KMS-26,

KMS28-BM, KMS-34, and MM.1S in this study. These were purchased from the Health Science

Research Resources Bank (Osaka, Japan), where cell line authenticity and Mycoplasma infection have been routinely checked by DNA fingerprinting and PCR. We used human bone marrow-derived stromal cell lines UBE6T-7 and stroma-NK, which were immortalized by transducing with a telomerase catalytic protein subunit, as BMSCs (5). Primary bone marrow cells were isolated from MM patients at the time of diagnostic procedure and used with or without positive selection of MM cells using CD138 MicroBeads and MACS separation columns (Miltenyi

Biotech, Gladbach, Germany). Normal plasma cells were isolated similarly from healthy volunteers (purchased from LONZA, Basel, Switzerland). Written informed consent was obtained in accordance with the Declaration of Helsinki and the protocol was approved by the

Institutional Review Board of Jichi Medical University.

In vitro co-culture system with BMSCs to recreate the BM microenvironment

We used a culture system devised by a modified cell culture insert to analyze the functional interaction between MM cells and BMSCs (5). First, BMSCs were cultured on the reverse side of the polyethylene terephthalate track-etched membrane of a high pore density cell culture insert

(35-3495; Becton-Dickinson, San Jose, CA) in a 24-well plate (35-3504; Becton-Dickinson).

After obtaining a confluent feeder layer, MM cells were seeded on the upper side of the membrane where the cytoplasmic villi of BMSCs pass through the etched 0.4-µm pores. In another set of conditions, MM cells were seeded on the upper side of a low pore density cell culture insert

(35-3095; Becton-Dickinson). Under this condition, MM cells were physically separated from the stromal layer, providing an adhesion-negative control. We performed the co-culture under

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hypoxic (5% O2) conditions to reproduce the BM microenvironment (20, 21).

Construction and production of lentiviral expression vectors

We used the lentiviral short-hairpin RNA/short-interfering RNA (shRNA/siRNA) expression vector pLL3.7 for knockdown experiments. Oligonucleotides containing siRNA target sequences are shown in Supplementary Table S1. Scrambled sequences were used as controls. We used the lentiviral vector CSII-CMV-MCS-IRES-VENUS (provided by Dr. Hiroyuki Miyoshi, RIKEN

BioResource Center, Ibaraki, Japan) containing the coding region of Ikaros cDNA for gain-of-function experiments. These vectors were co-transfected into 293FT cells with packaging plasmids (Invitrogen, Carlsbad, CA) to produce infective lentiviruses in culture supernatants. Lentiviruses were then added to cell suspensions in the presence of 8 µg/ml polybrene and transduced for 24 hours as previously described (5).

Reporter assays

We amplified the promoter regions of the CD180 gene (–1955 to +18, –1547 to +18, –1254 to +18,

-1040 to +18, and –384 to +18) using PCR and inserted them into the pGL4.17 firefly luciferase vector (Promega, Madison, WI) to generate reporter plasmids. A mutation was inserted at nucleotide position -1076 to -1070 and -1059 to -1052 by PCR-based site directed mutagenesis using wild-type reporter plasmid as a template (for primers, see Supplementary Table S2). We introduced reporter plasmids into KMS12-BM cells along with the pGL4.73 Renilla luciferase vector, which served as a positive control to determine transfection efficiencies, using electroporation. After 48 hours, firefly and Renilla luciferase activities were measured discriminately using the Dual-Luciferase Reporter Assay System (Promega). The promoterless pGL4.17-basic vector was used as a negative control. Luciferase activity was normalized with

KIKUCHI et al. the internal standard and indicated as a relative ratio to negative controls.

Chromatin immunoprecipitation assays

We used the ChIP-IT Chromatin Immunoprecipitation Kit (Active Motif, Carlsbad, CA) to perform chromatin immunoprecipitation assays. In brief, cells were fixed with 1% formaldehyde at 37 °C for 5 minutes, and sonicated to obtain chromatin suspensions. After centrifugation, supernatants were incubated with antibodies of interest at 4 °C overnight. The mixture was then incubated with protein A-agarose beads at 4 °C for 1 hour and centrifuged to collect the beads.

DNA fragments bound to the beads were purified with vigorous washing and subjected to PCR.

Detailed information on primers, including sequences, corresponding nucleotide positions, and

PCR product sizes, is shown in Supplementary Table S3.

Xenograft murine MM model

For ex vivo tracing of tumors, we inoculated a luciferase-expressing subline of RPMI8226 with or without BMSCs in NOD/SCID mice (Charles River Laboratories, Wilmington, MA).

Tumor-derived luciferase activity was measured by the IVIS Imaging System with Living Image software (Xenogen, Alameda, CA) (5). All animal studies were approved by the Institutional

Animal Ethics Committee and performed in accordance with the Guide for the Care and Use of

Laboratory Animals formulated by the National Academy of Sciences.

Other conventional techniques are described in Supplementary Materials and Methods.

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Results

The CD180/MD-1 complex is overexpressed in MM cells but not in normal plasma cells

In an initial effort to understand the role of CD180 in MM biology, we screened for the expression of TLRs in primary MM samples using the datasets in the Oncomine and GEO databases. We found a significant increase in transcription and DNA copy numbers of the CD180 gene in MM cells relative to normal plasma cells (PCs) (Figure 1A and Supplementary Figure S1A). CD180 mRNA expression was significantly up-regulated in CD138-negative stem-like MM cells

(Supplementary Figure S1B) and plasma cell leukemia (Supplementary Figure S1C). In contrast, no difference was observed in the expression of other TLRs between MM and normal PCs (Figure

1A). Previous studies showed that CD180 is a homologue of TLR4 and requires the accessory molecule MD-1 to appear on the cell surface and recognize LPS. In addition, CD180 requires

TLR4 or CD19 to transduce signals, because it lacks C-terminal Toll/interleukin-1 intracellular signaling domains (22-24). We therefore examined whether MM cells co-express MD-1 and

TLR4 with CD180 using CD138-positive cells isolated from the bone marrow of MM patients and healthy volunteers. As anticipated, most of primary MM cells expressed CD180 and MD-1 very strongly as well as TLR4 moderately compared with normal peripheral blood mononuclear cells, whereas normal plasma cells showed only faint expression by semi-quantitative RT-PCR (Figure

1B). Next, we isolated BM mononuclear cells from 6 MM patients and 2 healthy volunteers, and immediately stained them with specific antibodies against CD138, CD180, MD-1 and TLR4. We confirmed the co-expression of CD180 and MD-1 in CD138-positive myeloma cells but not in

CD138-negative non-myeloma BM cells or normal PCs (Figure 1C and Supplementary Figure

S2A). TLR4 was moderately expressed on CD138-positive MM cells (Figure 1C) and its co-expression with CD180 was detected in a minor fraction of cells (Supplementary Figure S2B).

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Similar expression patterns for CD180, MD-1 and TLR4 were obtained in 6 MM cell lines by

RT-PCR (Figure 1D, Top panel), flow cytometry (Figure 1D, Bottom panel and Supplementary

Figure S2B) and immunocytochemistry (Figure 1E). Overall, the CD180/MD-1 complex is highly and universally expressed in primary MM cells as well as MM cell lines. Notably, the signal intensity of CD180 is stronger in freshly isolated primary MM cells than MM cell lines, suggesting the positive impact of BM microenvironment on CD180 expression. In support of this view, the signal intensity of CD180 mRNA gradually declined after isolation from the bone marrow in cultured MM cells (Supplementary Figure S3A).

LPS enhances myeloma cell growth in positive correlation with the expression levels of CD180 in vitro

We reproduced the BM microenvironment, in which dormant MM cells physically interact with

BMSCs under hypoxic conditions, using a co-culture system to examine its regulatory effects on

CD180 expression. In brief, we cultured 3 MM cell lines (KMS12-BM, RPMI8226 and

KMS-21) with or without direct adhesion to BMSCs (UBE6T-7 and stroma-NK) in a cell culture insert under normoxic (20% O2) or hypoxic (5% O2) conditions. Real-time quantitative RT-PCR

(Q-PCR) analyses revealed that the expressions of both CD180 and MD-1 were markedly up-regulated via adhesion to BMSCs under hypoxic conditions (Figure 2A, Top panel and

Supplementary Figures S3B and S4A). This increase in mRNA expression resulted in the coordinated up-regulation of the surface expression of CD180 on MM cells (Figure 2A, Bottom panel). In addition, CD180 expression, with regard to both mRNA and surface expression, increased in a time-dependent manner under adherent and hypoxic conditions (Supplementary

Figure S4B). In contrast, no obvious changes were detectable in the expression of other TLRs, including TLR1, 2, 4, 5 and 6, in adherent MM cells (Supplementary Figure S4C). Therefore, it

KIKUCHI et al. is highly likely that CD180 is a marker of dormant MM cells localized in the BM microenvironment.

Functionally, the overexpressed CD180/MD-1 complex may sense LPS to activate dormant

MM cells, leading to regrowth and/or anti-apoptotic response. To test this possibility, we screened for the effects of various TLR ligands, such as LPS (for TLR4 and CD180), anti-CD180 antibody (for CD180), Pam3Csk (for TLR1 and 2) and flagellin (for TLR5), as well as poke weed mitogen (TLR-independent B-cell stimulant) as a control on myeloma cell growth (Figure 2B/C and Supplementary Figure S4D). Among these, LPS and an activating antibody against CD180

(18) significantly enhanced the growth of KMS12-BM and RPMI8226 cells in a dose-dependent manner under adherent and hypoxic conditions, which was positively correlated with the expression levels of the CD180/MD-1 complex (Supplementary Figure S4E). LPS failed to do so under non-adherent and normoxic conditions (Figure 2B, Normo). Moreover, a non-activating

CD180 antibody did not potentiate the growth of CD180-positive MM cells (Supplementary

Figure S4F) and poke weed mitogen activated MM cells irrespective of culture conditions

(Supplementary Figure S4D), indicating the specificity of CD180-mediated signal transduction leading to the proliferative response. Since TLR pathways activate several intracellular signaling elements, including extracellular signal-regulated kinase (ERK), p38 MAP kinase and c-Jun

N-terminal kinase (JNK) (9, 10), we examined their activation/phosphorylation status in

CD180-engaged MM cells. Immunofluorescent staining revealed the activation of JNK and ERK, which coincided with CD19 but not TLR4 expression, in CD180-positive MM cells after stimulation with either LPS or anti-CD180 antibody (Figure 2D/E and Supplementary Figure S4G).

These results strongly suggest that LPS enhances the growth of MM cells via the CD180/MD-1 pathway by the aid of CD19 (9, 24), which was expressed on dormant MM cells via the interaction

KIKUCHI et al. with BMSCs (25, 26).

LPS enhances myeloma cell growth in positive correlation with the expression levels of CD180 in vivo

Next, we determined the effects of LPS on myeloma cell growth in vivo. For this purpose, we used a murine xenograft model, in which human MM cells were inoculated with BMSCs in immunodeficient mice to recapitulate human MM-stroma interaction in the BM milieu (5). As illustrated in Figure 3A, a luciferase-expressing RPMI8226 subline (RPMI8226-Luc) and

UBE6T-7 stroma cells were either injected separately into the right and left thighs, respectively

(separate injection) or injected as a mixture into the right thigh (mixed injection) of immunodeficient mice. When tumors were measured by luciferase assay, either LPS (1 mg/kg) or vehicle (0.9% NaCl) was intraperitoneally administered twice a week for 3 weeks (n=3 in each group). We compared the tumor sizes between vehicle-control and LPS-administered groups on day 21. Tumor growth was significantly accelerated by LPS when MM cells were co-injected with BMSCs (mixed-LPS), whereas there was no difference in case of separate injection (Figure

3A/B). Histopathological examination also revealed tumor hypercellularity in mixed-injected mice administered with LPS, but not in the 3 other conditions (Figure 3C). CD180 expression was readily detected in MM cells co-injected with BMSCs (mixed/Control), but not in the case of separate injection, and CD180/CD138 double-positive cells were markedly increased by LPS administration (mixed/LPS) (Figure 3D). These results suggest that LPS induces clonal expansion of CD180-positive MM cells in vivo.

Next, we performed knockdown experiments to investigate whether LPS-triggered growth is mediated via the CD180 pathway in MM cells. We established RPMI8226-Luc sublines lentivirally transduced with shRNA against CD180 (sh-CD180) and an ineffective control

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(sh-control). We selected two sublines (sh-CD180#1 and #2) in which CD180 expression was significantly down-regulated relative to the control (Supplementary Figure S5A). CD180 silencing itself did not affect the proliferative potential or viability of myeloma cells (Figure 3E,

Control). First, we examined the effects of CD180 knockdown on LPS-triggered cell growth in vitro using the co-culture system. As anticipated, LPS-enhanced cell growth was almost completely abrogated by CD180 silencing under adherent and hypoxic conditions (Figure 3E,

LPS). We then attempted to confirm the effects of CD180 knockdown in vivo. The sh-control and sh-CD180 cells were inoculated with UBE6T-7 cells as a mixture in the right thigh of immunodeficient mice. When measurable tumors were detected by luciferase assay, either LPS or vehicle was intraperitoneally administered twice a week for 2 weeks (n=3 in each group). We compared the tumor sizes between vehicle-control and LPS-administered groups on day 18. LPS significantly enhanced the growth of sh-control sublines in mice, but not of CD180-knockdown sublines (Figure 3F/G). In previous studies, LPS facilitates BMSCs to produce inflammatory cytokines, such as interleukin-6, TNF- and IGF-1, which act in favor of MM cell growth (27, 28).

Therefore, it is possible that LPS indirectly enhances the growth of MM cells by stimulating cytokine production from stromal cells. To exclude this possibility, we confirmed the absence of

LPS receptors, TLR4 and CD180, in UBE6T-7 and stroma-NK cells using RT-PCR

(Supplementary Figure S5B). Moreover, the growth of RPMI8226 cells is known to be cytokine-independent (29). Taken together, we conclude that LPS directly enhances myeloma cell growth via the CD180/MD-1 pathway.

Regulation of CD180 promoter by IKZF1 transcription factor (Ikaros)

Next, we investigated the mechanisms of transcriptional activation of the CD180 gene via

KIKUCHI et al. myeloma-stroma interactions. First, we performed reporter assays to determine the regulatory elements in CD180 promoter. We subcloned 4 promoter fragments, 1955 to +18, 1254 to +18,

1040 to +18, and 384 to +18, into pGL4 luciferase vector to generate reporter plasmids (Figure

4A). The analyzed regions were selected by database search for CD180 promoter

(Supplementary Figure S6). Reporter assays revealed that deletion of the segment between

1254 and 1040 significantly decreased CD180 promoter activity (Figure 4B). This segment contains putative binding sites for the Ikaros family (IKZF), NF-B and C/EBPs. Among these, we focused on IKZF-binding sites because IKZF1 (Ikaros) and IKZF3 (Aiolos) are known to be critical activators for myeloma master oncogenes IRF4 and c-myc as well as target molecules of lenalidomide, one of the most effective drugs for MM (30, 31). We constructed mutant promoters carrying a non-binding mutation at the upstream IKZF-binding site (#1), the downstream IKZF-binding site (#2), and both (#1/#2) (Figure 4C). Promoter activities of the mutants #2 and #1/#2 were reduced to the background level in KMS12-BM cells, whereas the mutant #1 fully retained the activity (Figure 4D). To examine the involvement of NF-B in

CD180 regulation, we treated KMS12-BM cells, in which the classical NF-B pathway is constitutively activated for unidentified mechanisms (32), with a cell membrane-permeable peptide that inactivates NF-B activity by inhibiting nuclear translocation of p50 (17, 33). The inhibition of NF-B activity did not affect the expression of CD180 transcripts at all

(Supplementary Figure S7A). These results suggest that CD180 transactivation was mostly driven by Ikaros-family transcription factors through the downstream IKZF-binding site in the region between 1254 and 1040 of CD180 promoter.

To determine whether IKZF-family proteins are actually involved in CD180 transactivation, we first examined the expression of Ikaros and Aiolos in MM cells under adherent and hypoxic

KIKUCHI et al. conditions. Q-PCR and immunoblot analyses clearly showed that cell adhesion increased Ikaros expression but decreased Aiolos expression in MM cell lines under hypoxia (Figure 4E/F and

Supplementary Figure S7B for data quantification). In addition, we examined the status of EZH2 and its specific target site H3K27, because cell adhesion diminishes the abundance of H3K27me3 via EZH2 inactivation, leading to the transactivation of pro-survival genes in MM cells (5, 6). As anticipated, the methylation level of H3K27 was remarkably diminished in parallel with phosphorylation and down-regulation of EZH2 in MM cells under adherent and hypoxic conditions (Figure 4E/F and Supplementary Figure S7C). No difference was observed in other histone modifications including H3K4me3 and H3K9me3 under the same conditions

(Supplementary Figure S7C). From these results, we reasoned that H3K27 demethylation and subsequent recruitment of Ikaros at the downstream IKZF-binding site may promote CD180 transcription in MM cells in direct contact with BMSCs. To confirm this hypothesis, we performed chromatin immunoprecipitation (ChIP) assays. H3K27 was readily trimethylated at the downstream IKZF-binding site (Figure 4G, Top panel), reflecting a relatively low baseline expression of CD180 in MM cells without cell adhesion. H3K27 was completely demethylated when MM cells were adhered to BMSCs under hypoxic conditions. Ikaros binding became detectable at this site concomitantly with H2K27 demethylation (Figure 4G, Bottom panel).

Finally, we examined the effects of IKZF overexpression and knockdown on CD180 expression in

MM cells. Consistent with the results of ChIP assays, Ikaros overexpression caused an approximately 3-fold increase in CD180 mRNA abundance (Figure 4H), and reciprocally, its knockdown decreased CD180 expression to the same extent as Ikaros down-regulation (Figure 4I).

In contrast, the modulation of IKZF3 (Aiolos) expression levels did not affect the abundance of

CD180 expression in MM cells (Supplementary Figure S7D). These data define IKZF1 (Ikaros)

KIKUCHI et al. as a critical transactivator of the CD180 gene in MM cells.

IMiDs ameliorate LPS-triggered myeloma cell growth via down-regulation of CD180 expression

The above notion prompted us to speculate that lenalidomide and its analogous IMiDs, such as pomalidomide, ameliorate LPS-triggered myeloma cell growth by targeting CD180 due to their ability of inducing cereblon-dependent degradation of Ikaros in MM cells (30, 31). To substantiate this assumption, we examined whether the two IMiDs repressed CD180 expression in parallel with the reduction of Ikaros expression levels in MM cell lines co-cultured with BMSCs under hypoxia. Immunoblot analyses confirmed the dose-dependent decline in Ikaros expression by lenalidomide and pomalidomide (Figure 5A and Supplementary Figure S7E). With virtually identical kinetics, the two drugs reduced CD180 expression at the mRNA level and on the cell surface (Figure 5B and Supplementary Figure S7F). ChIP assays revealed that lenalidomide treatment readily decreased Ikaros binding to the IKZF-binding site of CD180 promoter with a reciprocal increase in H3K27 trimethylation in KMS12-BM cells (Figure 5C). In parallel with

CD180 suppression, lenalidomde treatment mitigated LPS-enhanced MM cell growth (Figure 5D).

Next, we verified the effects of lenalidomide on CD180 expression in primary MM cells. We isolated BM mononuclear cells from 4 MM patients and cultured them with or without lenalidomide for 24 hours. Flow cytometric analyses revealed that lenalidomide significantly down-regulated CD180 expression in CD138-positive MM fractions (Figure 5E/F). Taken together, IMiDs could suppress the LPS-triggered progression of MM by targeting CD180 expression.

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Lenalidomide ameliorates LPS-triggered myeloma cell growth in vivo

We attempted to confirm the inhibitory effect of lenalidomide on LPS-triggered myeloma cell growth in vivo. The mixture of RPMI8226-Luc and UBE6T-7 cells was inoculated into immunodeficient mice as described above. When measurable tumors developed, mice were randomly assigned to 4 groups (n=3 in each group) and treated with vehicle alone (0.3% DMSO in

0.9% NaCl), LPS alone (1 mg/kg, twice a week), lenalidomide alone (10 mg/kg, once a week), or both LPS and lenalidomide (single administration of lenalidomide followed by two injections of

LPS in a week) for 3 weeks (Figure 6A). The administration of LPS significantly enhanced tumor growth compared with vehicle control (Figure 6A/B). Lenalidomide alone failed to retard the growth of MM cells at the dose and schedule used in this experiment, but completely cancelled

LPS-induced growth enhancement in vivo. A histopathological examination confirmed the growth-promoting effect of LPS and its specific abrogation by lenalidomide (Figure 6C).

Immunofluorescent staining revealed a striking increase in CD138-positive cells along with

CD180-positive as well as double-positive cells in LPS-treated mice. These cells were completely eradicated from tumor regions by lenalidomide treatment (Figure 6D).

Discussion

In the present study, we show that a non-canonical TLR complex, CD180/MD-1, is specifically expressed on MM cells and senses bacterial LPS to transduce proliferative signals, leading to the regrowth of dormant MM clones. MM cells obtain clonal dormancy via VLA-4-mediated adhesion to BMSCs, which in turn phosphorylates and inactivates the H3K27 methyltransferase

EZH2 to derepress several genes conferring cell cycle arrest and drug resistance (5). We found that CD180 is an EZH2-regulated gene in MM cells and is transcriptionally activated by the

KIKUCHI et al. recruitment of Ikaros, an important transcription factor for hematopoietic stem cell maintenance and lymphocyte development (34), to CD180 promoter upon H3K27 demethylation (Figure 6E).

Furthermore, we demonstrated that lenalidomide and its analog pomalidomide ameliorate

LPS-triggered MM cell growth by silencing CD180 transcription to disrupt this circuit. In accordance with this model, disease progression occurred in 7 of 10 MM patients complicated with bacterial infections in our cohort (Supplementary Figure S8 and Table S4). Furthermore, post-infectious disease progression was not observed in 2 patients under maintenance therapy with lenalidomide or pomalidomide. Because the sample size is too small to draw a firm conclusion, we have started a prospective study to verify this concept in a bigger cohort.

The CD180/MD-1 complex shares the architecture and cell surface localization with the

TLR4/MD-2 complex, a canonical LPS receptor. The CD180/MD-1 complex has been proposed to play a role in fine-tuning of the cellular response to LPS, but there is still some controversy regarding its mechanisms (35). The CD180/MD-1 complex down-modulates the responses to

LPS through direct interaction with the TLR4/MD-2 complex in dendritic cells and macrophages

(36, 37), whereas LPS stimulates the proliferation of B cells via the CD180/MD-1 complex (22, 23,

38). The structural difference between MD-1 and MD-2 explains the low affinity of MD-1, which has a shallower cavity for direct binding to LPS (39). However, a recent crystallographic study demonstrates the structural basis for lipid and endotoxin binding to the CD180/MD-1 complex through a series of atomically detailed molecular simulations (40). The MD-1 cavity was expanded by a decrease in entropy to accommodate endotoxin binding, such that the

CD180/MD-1 complex acts as a sink and source of LPS for TLR4. In this scenario, the

CD180/MD-1 complex sequesters LPS from TLR4 to prevent overamplification of the TLR4 response, which ultimately leads to endotoxin shock (41). In MM cells, however, the

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CD180/MD-1 complex enhances the LPS response to stimulate cell proliferation because of the relatively lower expression of TLR4. This finding may increase our understanding of the functional diversity of TLRs.

From a mechanistic standpoint, our findings also provide insight into another enigmatic observation that the sensitivity of lenalidomide is positively correlated with the expression level of its target molecule Ikaros in MM cells (42, 43). We showed that Ikaros expression was increased in MM cells via adhesion to BMSCs under hypoxia. According to Jakubikova et al. (44), dormant MM cells, detected as SP-fraction cells by flow cytometry, are highly sensitive to lenalidomide especially in the presence of BMSCs. The causal link between dormancy and high

Ikaros expression is well explained by our findings. Hence, lenalidomide can selectively eradicate dormant MM cells with high Ikaros expression in the BM microenvironment (45). In addition, we have shown that the combination of bortezomib and lenalidomide exerts additive cytotoxicity against MM cells adhered to BMSCs, whereas the same combination was antagonistic under stroma-free conditions (46). Cell adhesion increases the cytotoxic activity of lenalidomide by elevating Ikaros expression, which in turn yields additive effects with other drugs.

Recent studies with next generation sequencing disclosed the complex genomic architecture of MM (47, 48). A model combining “Big Bang” dynamics and Darwinian type of evolution has been put forward to explain the development and progression of the disease (48). According to this model, a “Big Bang” leads to the early establishment of intra-tumoral heterogeneity, followed by Darwinian evolution to generate different subclones with additional abnormalities

(Supplementary Figure S9). There are multiple clones with variable abilities to propagate descendants at each step of disease progression. An increase in these reservoir clones may strongly affect the biological behavior of the disease, including malignant phenotype and drug

KIKUCHI et al. sensitivity. The entire process is strongly influenced by the interaction of each clone with the

BM microenvironment. The present study discloses a previously unknown factor to affect the disease process. Bacterial infection may act as a driving force of disease progression to accelerate clonal heterogeneity, ultimately leading to clonal dominance of the selected ones. In addition, our findings suggest an indispensable role of IMiDs in maintenance (49) and/or continuous therapies (50) to improve the treatment outcome by inhibiting infection-triggered disease progression along with depletion of myeloma stem cells.

Acknowledgments

The authors thank Dr. Akihiro Umezawa (National Research Institute for Child Health and

Development, Tokyo, Japan) and Dr. Hirofumi Hamada (Sapporo Medical University, Sapporo,

Japan) for providing UBE6T-7 and stroma-NK cell lines, respectively. We are grateful to Ms.

Akiko Yonekura and Ms. Michiko Ogawa for their technical assistance.

Grant Support

This work was supported in part by the High-Tech Research Center Project for Private

Universities: Matching Fund Subsidy from MEXT (to Y. Furukawa), and a Grant-in-Aid for

Scientific Research from JSPS (to J. Kikuchi, D. Koyama and Y. Furukawa). J. Kikuchi and Y.

Furukawa were funded by the Japan Leukemia Research Fund, Yasuda Memorial Cancer

Foundation, Takeda Science Foundation, and Novartis Foundation Japan. J. Kikuchi was also funded by Mitsui Life Social Welfare Foundation and SENSHIN Medical Research Foundation.

J. Kikuchi and Y. Furukawa received the Kano Foundation Research Grant and the Award in

KIKUCHI et al.

Aki’s Memory, respectively, from the International Myeloma Foundation Japan.

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Figure legends

Figure 1. The CD180/MD-1 complex is overexpressed in MM cells but not in normal plasma cells

[A] Messenger RNA expression of TLR1, 4, 5, 6, and CD180 in primary samples according to the

Oncomine database (http://www.oncomine.org). Normal, MGUS, and MM indicate

CD138-positive plasma cells derived from healthy volunteers (n=45), monoclonal gammopathy of undetermined significance (n=5), and multiple myeloma (n=131), respectively. *P <0.05 determined by one-way ANOVA with the Bonferroni post-hoc test. [B] Top panel:

Semi-quantitative RT-PCR analyses for TLR4, CD180, MD-1, and GAPDH (internal control) mRNA expression in CD138-positive cells derived from 10 MM patients, normal plasma cells

(PC) and peripheral blood mononuclear cells from a healthy volunteer (PBMNC). The results of suboptimal amplification cycles (40 cycles) are shown (see Supplementary Table S5 for primer sequences). Bottom panel: The signal intensities of each band were quantified, normalized to those of corresponding GAPDH, and are shown as relative values with those of PBMNC set at 1.0.

[C] Cytospin specimens of MM patient-derived BM mononuclear cells (MM#12 and #13) and normal plasma cells (PC#1 and #2) were stained with a combination of FITC-conjugated anti-CD138 and PE-conjugated anti-TLR4 antibodies, PE-conjugated anti-CD138 and

FITC-conjugated anti-CD180 antibodies, or FITC-conjugated anti-CD180 and Alexa Fluor

594-conjugated anti-MD1 antibodies. Nuclei were counterstained with DAPI. Arrows in each panel indicate cells double-positive for CD138 and TLR4, CD138 and CD180, or CD180 and

MD-1. [D] Top panel: Total cellular RNA was isolated from 6 MM cell lines and subjected to semi-quantitative RT-PCR analysis for the expression of TLR4, CD180, MD-1, and GAPDH

(internal control) mRNA. Bottom panel: Flow cytometric analysis of the expression of TLR4

KIKUCHI et al. and CD180 on 6 MM cell lines and PBMNC. The means ± S.D. (bars) of three independent experiments are shown. Representative bivariate dot-blots are depicted in Supplementary Figure

S2B. [E] Cytospin specimens of RPMI8226 cells were stained with a combination of

FITC-conjugated anti-CD180 with PE-conjugated anti-CD138, PE-conjugated anti-TLR4, or

Alexa Fluor 594-conjugated anti-MD1 antibody. Nuclei were counterstained with DAPI.

Figure 2. LPS enhances myeloma cell growth in positive correlation with the expression levels of CD180 in vitro.

[A] MM cell lines were cultured with or without adhesion (ad) to either UBE6T-7 or stroma-NK cells under normoxic (Normo) or hypoxic (Hypo) conditions in the co-culture system for 48 hours.

Top panel: CD180 mRNA expression by Q-PCR. Data were quantified by the 2–∆∆Ct method using GAPDH as a reference and are shown as fold increases against the values for cells cultured without adhesion under normoxic conditions (Normo). Bottom panel: The proportion of

CD180-positive cells was determined by flow cytometry. [B] MM cells were cultured in the presence of LPS with or without adhesion (ad) to BMSCs under normoxic (Normo) or hypoxic

(Hypo) conditions. Cell proliferation was assessed by the MTT assay after 72 hours and is shown as relative values of cells cultured without adhesion under normoxic conditions (Normo). [C]

RPMI8226 cells were cultured with either anti-CD180 antibody (MHR73-11) or the isotype-matched control (IgG1) under adherent and hypoxic conditions. Cell proliferation was assessed by the MTT assay after 72 hours and is shown as relative values of untreated cells. The means ± S.D. (bars) of 3-5 independent experiments. *P <0.05 by one-way ANOVA with Tukey’s multiple comparison test. [D] KMS12-BM and RPMI8226 cells were cultured for 48 hours with adhesion to UBE6T-7 cells under hypoxic conditions, followed by stimulation with LPS or anti-CD180 antibody for 1 hour. Cytospin specimens were stained with FITC-conjugated

KIKUCHI et al. anti-CD180 antibody and either anti-phosphorylated JNK [D] or anti-phosphorylated ERK [E], followed by staining with Alexa Fluor 594-conjugated anti-mouse IgG. Nuclei were counterstained with DAPI.

Figure 3. LPS enhances myeloma cell growth in positive correlation with the expression levels of CD180 in vivo.

[A] We subcutaneously inoculated 5  106 luciferase-expressing RPMI8226-Luc cells and 5  106

UBE6T-7 cells either separately in the right and left thighs, respectively (separate), or as a mixture in the right thigh (mixed) of NOD/SCID mice. Mice were intraperitoneally administered 1mg/kg

LPS or vehicle (0.9% NaCl) twice a week for 3 weeks. Treatments were started when inoculated tumors were measurable, defined as day 0. Representative photographs on day 0 and day 21 are shown (original magnification: 2). [B] Quantitative data of in vivo bioluminescence imaging on day 0 and day 21 expressed as photon units (photons/s). *P <0.05 against the control determined by one-way ANOVA with Tukey’s multiple comparison test (n=3). [C] Tumor sections were obtained from mice and subjected to hematoxylin-eosin staining. Scale bars indicate 100 µm and

20 µm (inset). [D] Tumor sections were stained with PE-conjugated anti-CD138 and

FITC-conjugated anti-CD180 antibodies. Nuclei were counterstained with DAPI. [E] RPMI8226 cells transduced with shRNA against CD180 (sh-CD180 #1 and #2) or an ineffective control

(sh-control) were cultured with adhesion to UBE6T-7 cells under hypoxic conditions in the absence or presence of LPS. Cell proliferation was assessed by the MTT assay after 72 hours and is expressed as relative values of the corresponding untreated controls. *P <0.05 against the values of corresponding untreated cells determined by one-way ANOVA with the

Student-Newman-Keuls multiple comparisons test (n=6). [F] We subcutaneously inoculated 5 

106 luciferase-expressing RPMI8226-Luc cells transduced with sh-CD180#2 (sh-CD180) or

KIKUCHI et al. sh-control (sh-control) and 5  106 UBE6T-7 cells as a mixture in the right thigh of NOD/SCID mice. Mice were intraperitoneally administered 1mg/kg LPS or vehicle (0.9% NaCl) twice a week for 2 weeks. Representative photographs on day 0 and day 18 are shown (original magnification:

2). [G] Quantitative data of in vivo bioluminescence imaging on day 0 and day 18 expressed as photon units (photons/s). *P <0.05 determined by one-way ANOVA with Tukey’s multiple comparison test (n=3).

Figure 4. Regulation of CD180 promoter by Ikaros transcription factor

[A] Schematic view of deletion fragments of the CD180 promoter used in luciferase assays.

Relative locations of the putative binding sites of transcription factors are approximated by the symbols shown in the box. [B] We transfected pGL4.17 plasmids containing CD180 promoter fragments into KMS12-BM cells along with the pGL4.73 control plasmid and cultured them under adherent and hypoxic conditions for 48 hours. CD180 promoter activity was calculated as firefly luciferase activities with those of an empty expression vector set at 1.0 after normalization of transfection efficiencies using Renilla luciferase activity. The means ± S.D. (bars) of 3-6 independent experiments are shown. *P <0.05 against the value of -1955 promoter determined by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. [C] Schematic view of CD180 promoter fragments with mutations at IKZF-binding sites. × indicates the sites inserted with mutations. The arrows indicate the region of PCR amplification in ChIP assays.

[D] CD180 promoter activity was determined as described in panel B. *P <0.05 against the value of wild-type promoter (WT) determined by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test (n=6). [E] Q-PCR analyses of the expression of Ikaros, Aiolos and

EZH2 in KMS12-BM and RPMI8226 cells cultured with or without adhesion to UBE6T-7 under

KIKUCHI et al. hypoxic conditions for 48 hours. [F] Whole cell lysates were prepared during the experiments described in panel E and subjected to immunoblotting for the indicated molecules. [G]

Chromatin suspensions were prepared from KMS12-BM cells in the experiments described in panel E and immunoprecipitated with anti-H3K27me3, anti-Ikaros, and isotype-matched (IgG) antibodies. The resulting precipitates were subjected to PCR to amplify the downstream

IKZF-binding site of CD180 promoter as shown in panel C. Representative data of 50 cycles are shown. Input indicates that PCR was performed with genomic DNA. [H] Q-PCR analyses for the expression of Ikaros and CD180 in RPMI8226 cells transfected with either the CSII-VENUS

(mock) or CSII-VENUS-Ikaros (Ikaros) lentiviral vector. [I] Q-PCR analyses for the expression of Ikaros and CD180 in KMS12-BM cells transfected with either the pLL3.7-sh-control

(sh-control) or pLL3.7-sh-Ikaros (sh-Ikaros) lentiviral vector. Data were quantified by the 2–∆∆Ct method using simultaneously amplified GAPDH as a reference and shown as fold increases against the sh-control.

Figure 5. Lenalidomide ameliorates LPS-induced myeloma cell growth via down-regulation of CD180 expression

[A] KMS12-BM and RPMI8226 cells were cultured in the absence or presence of lenalidomide at the indicated concentrations under adherent and hypoxic conditions and subjected to immunoblot analyses for the indicated molecules after 48 hours. [B] Top panel: Total cellular RNA was isolated during the experiments described in panel A and subjected to Q-PCR to determine CD180 expression. Bottom panel: Flow cytometric analysis of CD180 expression during the experiments described in panel A. *P <0.05 against untreated controls determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n=5). [C] Chromatin immunoprecipitation assays of lenalidomide-treated KMS12-BM cells performed as described in Figure 4G. [D]

KIKUCHI et al.

KMS12-BM and RPMI8226 cells were cultured in the absence or presence of LPS (100 ng/ml) and lenalidomide at the indicated concentrations under adherent and hypoxic conditions. Cell proliferation was assessed after 72 hours and is expressed as a percentage of the value for corresponding untreated cells. *P <0.05 between LPS (–) and LPS (+) samples at the same concentration of lenalidomide determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n=5). [E] Flow cytometric analysis of CD180 expression in

CD138-positive BM fractions from MM patients after culture with or without lenalidomide for 24 hours. [F] The means ± S.D. (bars) of 4 independent experiments. *P <0.05 determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test.

Figure 6. Lenalidomide ameliorates LPS-induced myeloma cell growth in vivo

[A] We subcutaneously inoculated 5  106 luciferase-expressing RPMI8226-Luc cells and 5  106

UBE6T-7 cells as a mixture in the right thigh of NOD/SCID mice. Mice were intraperitoneally administered vehicle (0.3% DMSO in 0.9% NaCl), 1 mg/kg LPS alone, 10 mg/kg lenalidomide alone, or LPS plus lenalidomide at the indicated time schedule (arrows on top). In vivo luciferase activity was measured on days 7, 14, and 21. *P <0.05 determined by one-way ANOVA with

Tukey’s multiple comparison test (n=3). [B] Representative photographs on days 0 and 21

(original magnification: 2). [C] Hematoxylin-eosin staining of tumor sections. Scale bars indicate 100 µm and 20 µm (inset). [D] Tumor sections were stained with PE-conjugated anti-CD138 and FITC-conjugated anti-CD180 antibodies. Nuclei were counterstained with

DAPI. [E] Graphic abstract: Role of the CD180/MD-1 pathway in LPS-triggered myeloma cell growth and its therapeutic intervention with lenalidomide.

Figure 1 A TLR1 TLR4 TLR5 TLR6 CD180

centered intensity -

* log2 log2 median Normal MGUS MM Normal MGUS MM Normal MGUS MM Normal MGUS MM Normal MGUS MM

B C TLR4 [CD138/TLR4] [CD180/CD138] [CD180/MD-1]

CD180

MD-1

GAPDH #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 PC

MM patient CD138-positive cells MM#12 MM#13 PBMNC

15 TLR4 CD180

10 PC #1 #1 PC

5 2 relative intensity relative PC # PC

0 20 μm #1 #2 #3 #4 #5 #6 #7 #8 #9 #10PC PBMNC D E TLR4 Nuclei CD138 CD180 merge

CD180

MD-1

GAPDH Nuclei MD-1 CD180 merge 30

TLR4 CD180

10 Nuclei TLR4 CD138 merge positive cells (%) cells positive 0

30 μm Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2 A 15 3 40

Normo Normo+ad 10 30 2 Hypo CD180 Hypo+ad 20 5 1 10 relative relative mRNA expression mRNA 0 0 0 UBE6T-7 stroma-NK UBE6T-7 stroma-NK UBE6T-7 stroma-NK 20 20 ** 40 * * * 15 15 * 30 * *

positive positive * - 10 20 10 cells (%) cells 5 10 5 CD180 0 0 0 UBE6T-7 stroma-NK UBE6T-7 stroma-NK UBE6T-7 stroma-NK KMS12-BM RPMI8226 KMS21

B Normo Normo+ad Hypo Hypo+ad C * IgG anti-CD180

2 2.5 * * * * * 1.5 * * 2 *

1.5 relative cell number cell relative 1

relative cell number cell relative 1 0.5 LPS 50 100 50 100 50 100 50 100 (ng/ml) KMS12-BM RPMI8226 KMS12-BM RPMI8226 0.5 0 1 2 UBE6T-7 stroma-NK IgG/anti-CD180 (μg/ml) D E Control LPS anti-CD180 Control LPS anti-CD180

CD180/pJNK/DAPI CD180/pERK/DAPI BM BM - - KMS12 KMS12 RPMI8226 RPMI8226 RPMI8226 RPMI8226

30 μm 30 μm Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3 A Day 0 Day 21 B

sepa-control sepa-LPS [separate- mix-control mix-LPS control] 750 70 *

[separate- 2 LPS] 50 500 UBE6T7 RPMI8226-Luc /sec/cm p

photons/s) photons/s) 4

[mixed- 30 10

control] ×

250

( Luciferase activities activities Luciferase 10 mixed separate separate mixed [mixed-LPS] 0 UBE6T7 + RPMI8226-Luc Day 0 Day 21

C [separate-control] [separate-LPS] D

Nuclei CD138 CD180 merge

100 μm 20 μm 100 μm 20 μm

[mixed-control] [mixed-LPS]

100 μm 20 μm 100 μm 20 μm E control LPS 50 ng/ml LPS 100 ng/ml

1.6 [separate] [mixed]

* 1.4 Control LPS Control LPS * 20 μm number 1.2

relative cell relative 0.8 sh-control sh-control+LPS 0.6 G sh-control sh-CD180#1 sh-CD180#2 shCD180 shCD180+LPS

250 F Day 0 Day 18 *

200 [sh-control] 70

sr /

2 [sh-control 150 50

+ LPS] photons/s)

10 p/sec/cm

100

30 × ( 10 Luciferase activities activities Luciferase [sh-CD180]

10 [sh-CD180 + LPS] 0 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Day 0 Day 18 Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B Figure 4 Luc −1955 −1955 −1254 −1254 Luc

−1040 −1040 Luc * −384 Luc −384 * Luc Control IKZF NF-κB C/EBP XBP-1 0 5 10 15 C D relative luciferase activity [Control] Luc Control −1254 [WT] Luc WT

[Muta-#1] × Luc Muta-#1

[Muta-#2] × Luc Muta-#2 *

[Muta-#1/#2] ×× Luc Muta-#1/#2 * 0 2 4 6 8 IKZF NF-κB C/EBP relative luciferase activity

E F EZH2 3 4 adhesion (−) H3K27me3

adhesion (+)

3 2 Ikaros

2 Aiolos

expression

relative mRNA relative 1 1 Histone H3

GAPDH 0 0 Ikaros Aiolos EZH2 Ikaros Aiolos EZH2 adhesion (−) (+) (−) (+)

KMS12-BM RPMI8226 KMS12-BM RPMI8226

G H mock Ikaros I sh-control sh-Ikaros

500bp 14

12 1 adhesion (−) (+) (−) (+) (−) (+) 10

H3K27me3 IgG INPUT 8

6 0.5 500bp 4

adhesion (−) (+) (−) (+) (−) (+) 2 expression mRNA relative relative mRNA expression mRNA relative 0 0 Ikaros IgG INPUT Downloaded from cancerres.aacrjournals.org on SeptemberIkaros 24, 2021.CD180 © 2018 American AssociationIkaros for CD180Cancer Research. Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B Figure 5 KMS12-BM RPMI8226 1.5 CRBN

BM 1 - Ikaros 0.5 KMS12

GAPDH CD180 relative mRNA expression mRNA 0 0 1 2.5 5 CRBN 20 * *

Ikaros positive - 10 RPMI8226 cells (%) cells * * GAPDH * CD180 0 1 2.5 5 0 Lenalidomide (μM) 0 1 2.5 5 Lenalidomide (μM) C D LPS (−) * 500bp LPS (+) 150 150 *

Lena (−) (+) (−) (+) (−) (+) 125 125

H3K27me3 IgG INPUT 100 100

500bp relative cell number (%) number cell relative 75 75

Lena (−) (+) (−) (+) (−) (+) 50 50 Lena 0 1 2.5 5 0 1 2.5 5 Ikaros IgG INPUT (μM) KMS12-BM RPMI2886

E #16 #17 F 40 60 30 matched

- 20 control 10 cells)

Isotype *

0 cells 40 50

30 positive positive 51.6%51.6% 59.2% 59.2% -

0 20 positive -

(μM) 10 CD138 40

0 CD180 40 (% in (% in 30 34.5% 49.8% 2.5 34.5% 49.8% 20 Lenalidomide Lenalidomide

10 30 0 2.5 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 Lenalidomide (μM) Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer CD180-FITC Research. Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B Figure 6

[Lena] [Control] [LPS] [LPS]

2000 * 70

control sr /

2 1500 LPS 50

Lena

p/sec/cm [Lena] [Lena+LPS] 1000 5 photons/s)

30 5

Lena+L 10

#1 #3 #2 #1 #2 #3 #1 PS #1 #2 #3 #1 ×

(x 10 (x Luciferase activity Luciferase

500 2 2 10 #3 # #3 #3 # #3

0 0 7 14 21 Day 0 7 14 21 0 7 14 21 Time (days)

C D [Control] [LPS]

[Control] [LPS] CD180 /CD138/DAPI

100 μm 20 μm 100 μm 20 μm [Lena] [Lena+LPS] [Lena] [Lena+LPS]

100 μm 20 μm 100 μm 20 μm

20 μm

E Under hypoxia Lenalidomide LPS

TLR4 Ikaros CD180 H3K27me3 JNK nucleus Disease ERK progression

cell growth VLA-4 Myeloma cell

VCAM-1 fibronectin Stroma cell Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 23, 2018; DOI: 10.1158/0008-5472.CAN-17-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Myeloma Cells are Activated in Bone Marrow Microenvironment by the CD180/MD-1 Complex which Senses Lipopolysaccharide

Jiro Kikuchi, Yoshiaki Kuroda, Daisuke Koyama, et al.

Cancer Res Published OnlineFirst January 23, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-2446

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2018/01/23/0008-5472.CAN-17-2446.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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