MicroRNAs (miRNAs and miRs) are small (19–25 base pairs) non-coding RNAs with the ability to modulate gene expression. Previously, we showed that the miR-34 family is downregulated in multiple myeloma (MM) as the cancer progressed. In this study, we aimed to clarify the mechanism of miRNA dysregulation in MM. We focused particularly on the interaction between MYC and the TP53-miR34 axis because there is a discrepancy between increased TP53 and decreased miR-34 expressions in MM. Using the nutlin-3 or Tet-on systems, we caused wild-type (WT) p53 protein accumulation in human MM cell lines (HMCLs) and observed upregulated miR-34 expression. Next, we found that treatment with an Myc inhibitor alone did not affect miR-34 expression levels, but when it was coupled with p53 accumulation, miR-34 expression increased. In contrast, forced MYC activation by the MYC-ER system reduced nutlin-3-induced miR-34 expression. We also observed that TP53 and MYC were negatively correlated with mature miR-34 expressions in the plasma cells of patients with MM. Our results suggest that MYC participates in the suppression of p53-dependent miRNA expressions. Because miRNA expression suppresses tumors, its inhibition leads to MM development and malignant transformation.

Tissue inhibitors of metalloproteinases (TIMPs) are endogenous matrix metalloproteinase inhibitors. TIMP1 is produced by cancer cells and has pleiotropic activities. However, its role and source in multiple myeloma (MM) are unclear. Here, we evaluated TIMP1 protein and mRNA levels in bone marrow (BM) plasma cells and assessed the effects of TIMP1 expression on fibroblast invasive capacity using three-dimensional spheroid cell invasion assays. TIMP1 mRNA and protein levels were elevated when patients progressed from monoclonal gammopathy of undetermined significance or smouldering myeloma to MM. Furthermore, TIMP1 levels decreased at complete response and TIMP1 protein levels increased with higher international staging. TIMP1 mRNA levels were markedly higher in extramedullary plasmacytoma and MM with t(4;14). Overall survival and post-progression survival were significantly lower in MM patients with high TIMP1 protein. Recombinant TIMP1 did not directly affect MM cells but enhanced the invasive capacity of fibroblasts; this effect was suppressed by treatment with anti-TIMP1 antibodies. Fibroblasts supported myeloma cell invasion and expansion in extracellular matrix. Overall, these results suggested that MM-derived TIMP1 induces the invasive phenotype in fibroblasts and is involved in disease progression. Further studies are required to elucidate the specific roles of TIMP1 in MM and facilitate the development of novel therapies targeting the TIMP1 pathway.

Human endogenous retroviruses (HERVs) are retrotransposons that infect human germline cells and occupy 5-8% of the human genome. Their expression, though inhibited by mutation, deletion, and epigenetic mechanisms under normal conditions, is associated with diseases including cancer. This study aimed to clarify the association between HERVs and multiple myeloma (MM) progression. We found that HERV-K envelope (env) and long-term repeat (LTR) expression was statistically significantly higher within plasma cells in MM than in monoclonal gammopathy of undetermined significance or controls. HERV-K env knockdown increased proliferation in the MM.1S cell line and decreased the expression of the tumor suppressor genes TP53 and CDKN1A. TP53 and CDKN1A were highly expressed in MM, and their expression was correlated with HERV-K expression. HERV-K knockdown reduced apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3F, 3G, and 3H expression by 10-20% in MM.1S cells. The anti-retroviral agents nevirapine and nelfinavir suppressed proliferation and increased HERV-K expression in MM cell lines. Our results suggest that HERV-K is involved in MM progression, but its role is likely to go beyond promoting cell proliferation. Clarifying the role of HERV-K in MM will lead to the discovery of novel treatment strategies and supply new insights into MM pathogenesis.

Long noncoding RNAs (lncRNAs) are deregulated in human cancers and are associated with disease progression. Plasmacytoma Variant Translocation 1 (PVT1), a lncRNA, is located adjacent to the gene MYC, which has been linked to multiple myeloma (MM). PVT1 is expressed in MM and is associated with carcinogenesis. However, its role and regulation remain uncertain. We examined PVT1/MYC expression using real-time PCR in plasma cells purified from 59 monoclonal gammopathy of undetermined significance (MGUS) and 140 MM patients. The MM cell lines KMS11, KMS12PE, OPM2, and RPMI8226 were treated with JQ1, an MYC super-enhancer inhibitor, or MYC inhibitor 10058-F4. The expression levels of PVT1 and MYC were significantly higher in MM than in MGUS (p < 0.0001) and were positively correlated with disease progression (r = 0.394, p < 0.0001). JQ1 inhibited cell proliferation and decreased the expression levels of MYC and PVT1. However, 10054-F4 did not alter the expression level of PVT1. The positive correlation between MYC and PVT1 in patients, the synchronous downregulation of MYC and PVT1 by JQ1, and the lack of effect of the MYC inhibitor on PVT1 expression suggest that the expression of these two genes is co-regulated by a super-enhancer. Cooperative effects between these two genes may contribute to MM pathogenesis and progression.

Background: DNA polymerases (DNA pols) are essential enzymes for DNA replication. In mammalian cells, DNA pols are divided into four families: A (Pol θ, Pol γ, and Pol ν), B (Pol α, Pol δ, Pol ε, and Pol ζ), X (Pol β, Pol λ, Pol μ, and TDT), and Y (Pol η, Pol ι, Pol κ, and REV1). These DNA pols are required for both genome duplication and protecting cells from DNA damage induced by endogenous and exogenous agents, such as ROS, UV, and chemotherapeutic drugs. For example, Pol β, Pol λ, and Pol ι participate in base excision repair. Contrastingly, Pol ζ, REV1, Pol η, Pol ι, and Pol κ can replicate over various DNA lesions to prevent DNA replication stalling, known as translesion synthesis. Although some DNA pols are highly expressed in cancer cells, indicating chemotherapeutic resistance and poor outcome, their exact roles and expression mechanisms have not been fully elucidated. Multiple myeloma (MM) is a hematological malignancy of terminally differentiated plasma cells, with multistep progression from pre-cancer stage namely. In this study we attempted to elucidate the involvement of DNA pols in multistep oncogenesis of MM. Methods: A total of 63 MM and 29 MGUS patients, 15 controls, and 9 MM cell lines were included in the study. RNA was extracted from purified CD138+ plasma cells. DNA pol expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Doxycycline-inducible p53 system (Tet-on p53) and nutlin-3 were used for analyzing the role of p53 in DNA pol expressions in MM cell lines. Melphalan, doxorubicin, and bortezomib were used to examine DNA pol expressions in damaged cells in vitro. JQ1 and CPI203 were used to evaluate the role of bromodomain in DNA pol expressions. Results: Pol α and Pol ε expressions were significantly higher in MM than in control (p=0.007 and p=0.004, respectively), but Pol ε and Pol ζ levels were not significantly different (p=0.631, p=0.0826, respectively). Pol η, REV1, Pol ι, and Pol κ expressions were significantly higher in MM than control (p<0.001, p=0.002, p<0.001, and p<0.001, respectively). Pol θ and Pol γ were expressed at a higher level in MM than in control (p<0.001 and p<0.001, respectively). Pol β and Pol λ expressions were higher in MM than in control (p=0.0088 and p=0.013, respectively). Although the expressions of many DNA pols were higher in MM plasma cells, we focused on Pol η and Pol θ, because Pol λ, Pol μ, Pol ν, and Pol ι were expressed at very low levels, and Pol ε, Pol ζ, Pol γ, Pol κ, and REV1 were expressed in PBMNCs of healthy volunteers at high level. Pol η and Pol θ expressions did not differ due to known risk factors, such as cytogenetic abnormalities and ISS. Pol η expressions were positively correlated with p53 and myc expressions (r=0.718, p<0.001, r=0.528, p<0.001 respectively). p53 overexpression by Tet-on vector or nutlin-3 treatment enhanced Pol η expression, indicating that Pol η expression is regulated by p53. Melphalan or doxorubicin increased Pol η expression, but bortezomib or lenalidomide did not, suggesting that Pol η is upregulated by DNA damage via p53 pathway. Overall survival of the patients with high Pol η expression tended to be worse than with low Pol η expression (24 months survival: 69.6% vs. 57.9%, p=0.29). Pol θ expression was weakly correlated with p53. Melphalan induced Pol θ expression but doxorubicin did not. JQ1 significantly reduced Pol θ expression suggesting that Pol θ was regulated by bromodomain. Conclusion: We found that Pol θ and Pol η are highly expressed in MM, and upregulated by DNA damage. These DNA pols are involved in drug resistance and genomic instability leading to poor prognosis. Thus, DNA pols can be used as novel therapeutic targets and prognostic markers. Disclosures Handa: Ono: Research Funding.

Background: Chromosomal abnormalities are strongly associated with prognosis of multiple myeloma (MM). Among them, t (14;16) resulting in high expression of c-MAF, and t (14;20) resulting in high expression of MAFB, lead to poor prognosis. However, clinical significance and mechanisms underlying high c-MAF and MAFB expression without these translocations have not been fully elucidated. Methods: A total of 96 MM and 38 MGUS patients, 10 controls, and 9 MM cell lines were included in this study. RNA was extracted from purified CD138+ plasma cells from bone marrow (BM) mononuclear cells. c-MAF, MAFB, p53, and p21 RNA expressions were determined by RQ-PCR. Their expression levels were normalized against ACTB levels and calculated with 2-ΔΔCt value. Inhibition studies using BRD4 inhibitor JQ1, and MYC inhibitor10058-F4, and MM cell lines expressing doxycycline-inducible p53 (Tet-on p53) or c-MAF knockdown (KD) with siRNA were used for in vitro studies. Results: c-MAF expression in MM patients was higher than in MGUS patients and control (median level 0.043, 0.025, 0.002, p<0.001), but MAFB expression did not differ between MM, MGUS, and control (p=0.371). Although c-MAF expression level was significantly higher in MM with t (14;16) (p=0.0018), high c-MAF expression was observed in patients without the translocations, suggesting a role of c-MAF in MM progression. Other cytogenetic abnormalities such as del 17p, or t (4;14), did not affect c-MAF expression. Overall survival (OS) of MM patients with high c-MAF expression was significantly inferior compared to the patients with low c-MAF expression (HR 2.46, p=0.002, 2 years survival rate 42.9% vs. 72.7%, median 20.3 months vs. not reached), but progression-free survival (PFS) did not differ (p=0.551). Instead, post-progression survival (PPS) was significantly shorter in the patients with high c-MAF expression (HR 4.67, p<0.001, two years survival 0% vs. 60.2%, median 3.6 months vs. 49.3 months) suggesting that c-MAF confers drug resistance to residual disease. MAFB expression did not affect OS, PFS, and PPS. Reduction of cyclin D2 expression by c-MAF KD in vitro and a positive correlation between c-MAF and cyclin D2 expression (r=0.29, p<0.001) in vivo in patients, support previous reports describing that MM proliferation is induced by c-MAF via cyclin D2. c-MAF was not correlated with APOBEC3B expression. c-MAF expression was positively correlated with MDM2 and MYC (r=0.387, p=0.03 and r=0.221, p=0.019, respectively) but not with p53 and PUMA. A tendency of positive correlation was also observed with p21/CDK1NA (r=0.304, p=0.085). p53 overexpression from the Tet-on p53 and p53 stabilization by nutlin-3 increased c-MAF expression in three cell lines with t (14;16). c-MAF KD together with p53 overexpression significantly suppressed the proliferation, implying synthetic lethality. These findings suggest that c-MAF expression is upregulated in response to proliferation arrest induced by the p53 pathway; suppressing this responsive c-MAF expression abrogates cell growth more efficiently. JQ1 and CPI203 increased c-MAF expression in cell lines with t (14;16), suggesting that c-MAF expression is not controlled by super-enhancers. Conclusion: Although the mechanism of high expression of c-MAF and MAFB has previously been reported in IgH translocation, the relationship between c-MAF and drug resistance remains to be determined. The induction of c-MAF expression by tumor suppressor gene p53 is suggested to be a mechanism underlying poor prognosis of MM with t (14;16), and suppressing c-MAF expression with chemotherapeutic drugs might prevent emergence of drug resistance in residual tumor cells. Disclosures Handa: Ono: Research Funding.

Backgrounds and Aims: Matrix metalloproteinase (MMP) is endopeptidase enzyme degrading extracellular matrix, and tissue inhibitor of metalloproteinases (TIMP) is negative regulator of MMP. MMP is well known to be involved in metastatic mechanism of cancer cell and oncogenesis. However expression and role of MMP and TIMP has not been well established in multiple myeloma (MM). Therefore we examined whether expression of MMP and TIMP was involved in progression and prognosis of MM and extramedullary plasmacytoma (EMP) formation.

Background and Aims: Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes, such as proliferation, apoptosis, and cellular damage response. Long non-coding RNA (lnc RNA), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNA are deregulated in diverse human cancers and associated with disease progression, however little is available in multiple myeloma (MM). We have previously shown that lnc RNA MALAT1 was a stress response gene associated with MM progression. We found that lnc RNA NEAT1 is also highly expressed in MM cells by transcriptome analysis with next generation sequencer (NGS). NEAT1 is recently revealed to play an important role on DNA damage response (DDR) as downstream of p53, and thereby involves in carcinogenesis. However its exact role in cancers is still in controversy. In this study, we tried to elucidate role and regulation mechanism of NEAT1 during MM progression.

Background: Special region called superenhancer where Bromodomain-containing protein 4 (BRD4) complex densely accumulates attracts attention. MYC is a transcription factor highly expressing in many types of cancer and plays an important role on carcinogenesis. MYC possesses superenhancer and its expression is inhibited by BRD4 inhibitor JQ1. Recent transcriptome-wide analyses have revealed an overwhelming amount of transcribed but not translated non-coding RNAs capable of influencing diverse cellular processes such as proliferation, apoptosis, and motility. Long non-coding RNA (lnc RNAs), which are commonly defined as transcripts >200 nt in length, have emerged as a class of key regulatory RNA. LncRNAs are deregulated in diverse human cancers and associated with disease progression; however little is available in multiple myeloma (MM). LncRNA PVT1 locates adjacent to MYC is also reported to be associated with carcinogenesis and abnormal PVT1 fusing NBEA or WWOX in MM with 8q24 abnormality has been reported, but its roles and regulation machinery remain uncertain.