Erik Wendlandt

Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor β (TGF-β) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-β signaling, and its expression is induced by TGF-β. To confirm the relationship between miR-21 and the TGF-β signaling pathway, we measured the effect of miR-21 on a TGF-β-responsive reporter. We found that miR-21 enhanced TGF-β signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-β signaling, leading to increased fibrogenesis.

The role of microRNAs miR-200b and miR-200c in TLR4 signaling and NF-κB activation.

Recognition of microbial products by members of the Toll-like receptor (TLR) family initiates intracellular signaling cascades that result in NF-κB activation and subsequent production of inflammatory cytokines. We explored the potential roles of microRNAs (miRNAs) in regulating TLR pathways. A target analysis approach to the TLR4 pathway adaptor molecules identified several putative targets of miR-200a, miR-200b and miR-200c. miRNA mimics were co-transfected with a NF-κB activity reporter plasmid into HEK293 cells stably expressing TLR4 (HEK293-TLR4). Mimics of both miR-200b and miR-200c, but not miR-200a, decreased NF-κB reporter activity in either untreated cells or in cells treated with endotoxin:MD2 as a TLR4 agonist. Transfection of HEK293-TLR4 cells with miR-200b or miR-200c significantly decreased expression of MyD88, whereas TLR4, IRAK-1 and TRAF-6 mRNAs were unaffected. When miR-200b or miR-200c mimics were transfected into the differentiated monocytic THP-1 cell line, the abundance of MyD88 transcripts, as well as LPS-induced expression of the pro-inflammatory molecules IL-6, CXCL9 and TNF-α were diminished. These data define miRNAs miR-200b and miR-200c as factors that modify the efficiency of TLR4 signaling through the MyD88-dependent pathway and can thus affect host innate defenses against microbial pathogens.

Low serum miR-19a expression as a novel poor prognostic indicator in multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the clonal expansion of plasma cells. Despite continuing advances, novel biomarkers are needed for diagnosis and prognosis of MM. In our study, we characterized the diagnostic and prognostic potential of circulating microRNAs (miRNAs) in MM. Serum miRNA levels were analyzed in 108 newly diagnosed symptomatic MM patients and 56 healthy donors (HDs). Our analysis identified 95 dysregulated miRNAs in newly diagnosed MM patients. Of the 95 dysregulated miRNAs, dysregulation of miR‐19a, miR‐92a, miR‐214‐3p, miR‐135b‐5p, miR‐4254, miR‐3658 and miR‐33b was confirmed by quantitative reverse transcription PCR (RT‐qPCR). Receiver operating characteristic analysis revealed that a combination of miR‐19a and miR‐4254 can distinguish MM from HD with a sensitivity of 91.7% and specificity of 90.5%. Decreased expression of miR‐19a was positively correlated with international staging system advancement, del(13q14) and 1q21 amplification. Furthermore, downregulation of miR‐19a resulted in significantly decreased progression‐free survival (PFS) and overall survival (OS). Our analysis indicated that the poor prognostic correlation of miR‐19a expression was independent of genetic abnormalities in MM. Multivariate analysis revealed that miR‐19a was a significant predictor of shortened PFS and OS. Interestingly, although miR‐19a levels portend a poor prognosis, patients with low miR‐19a levels had an improved response to bortezomib compared to those with high miR‐19a profile. Patients with downregulated miR‐19a experienced a significantly extended survival upon bortezomib‐based therapy. These data demonstrate that the expression patterns of serum microRNAs are altered in MM, and miR‐19a levels are a valuable prognostic marker to identify high‐risk MM.

Bruton Tyrosine Kinase Is a Therapeutic Target in Stem-like Cells from Multiple Myeloma

Ibrutinib (Imbruvica), a small-drug inhibitor of Bruton tyrosine kinase (BTK), is currently undergoing clinical testing in patients with multiple myeloma, yet important questions on the role of BTK in myeloma biology and treatment are outstanding. Using flow-sorted side population cells from human myeloma cell lines and multiple myeloma primary samples as surrogate for the elusive multiple myeloma stem cell, we found that elevated expression of BTK in myeloma cells leads to AKT/WNT/β-catenin-dependent upregulation of key stemness genes (OCT4, SOX2, NANOG, and MYC) and enhanced self-renewal. Enforced transgenic expression of BTK in myeloma cells increased features of cancer stemness, including clonogenicity and resistance to widely used myeloma drugs, whereas inducible knockdown of BTK abolished them. Furthermore, overexpression of BTK in myeloma cells promoted tumor growth in laboratory mice and rendered side population-derived tumors that contained high levels of BTK more sensitive to the selective, second-generation BTK inhibitor, CGI1746, than side population-derived tumors that harbored low levels of BTK. Taken together, these findings implicate BTK as a positive regulator of myeloma stemness and provide additional support for the clinical testing of BTK-targeted therapies in patients with myeloma.

Copper–zinc superoxide dismutase-mediated redox regulation of bortezomib resistance in multiple myeloma

Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper–zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity.

Cancer stem cells are the cause of drug resistance in multiple myeloma: fact or fiction?

Multiple myeloma (MM) remains a largely incurable, genetically heterogeneous plasma-cell malignancy that contains – just like many other cancers – a small fraction of clonogenic stem cell-like cells that exhibit pronounced self-renewal and differentiation capacities, but also pronounced drug resistance. These MM stem cells (MMSCs) are a controversial but highly significant issue in myeloma research because, in our opinion, they are at the root of the failure of anti-neoplastic chemotherapies to transform myeloma to a manageable chronic disease. Several markers including CD138−, ALDH1+ and SP have been used to identify MMSCs; however, no single marker is reliable for the isolation of MMSC. Nonetheless, it is now known that MMSCs depend on self-renewal and pro-survival pathways, such as AKT, Wnt/β-catenin, Notch and Hedgehog, which can be targeted with novel drugs that have shown promise in pre-clinical and clinical trials. Here, we review the pathways of myeloma “stemness”, the interactions with the bone marrow microenvironment that promote drug resistance, and the obstacles that must be overcome to eradicate MMSCs and make myeloma a curable disease.

Junctional adhesion molecule-A is overexpressed in advanced multiple myeloma and determines response to oncolytic reovirus

Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.

Nek2 is a novel regulator of B cell development and immunological response.

The serine/threonine kinase Nek2 is commonly found upregulated in a wide variety of neoplasms including diffuse large B cell lymphoma and multiple myeloma. High expression of Nek2 is implicated in the induction of chromosomal instability, promotion of cell proliferation, and drug resistance in tumor cells as well as a marker for poor clinical outcomes. Despite its well recorded involvement in chromosomal instability and neoplastic growth, little is known about the involvement of Nek2 in B cell development. Here we report the development of a transgenic mouse line with conditional expression of Nek2 in the B cell lineage and the effects it has on the development of B cells. Interestingly, we found that the overexpression of Nek2 does not induce spontaneous tumor formation within the transgenic mice up to 24 months after induction. Instead, overexpression of Nek2 in the B cell lineage affects the development of B cells by increasing the proportion of immature B cells in the bone marrow and decreasing B-1 B cells in peritoneal cavity. Furthermore, Nek2 transgenic mice develop spontaneous germinal centers and exhibit an enhanced T cell dependent immune response. Altogether, our data demonstrates a novel role for Nek2 in regulating B cell development and the immune response.

Neoplastic plasma cell aberrant antigen expression patterns and their association with genetic abnormalities

Abstract Little is known about aberrant antigen expression patterns and their association with cytogenetic aberrations in multiple myeloma (MM). We examined the correlation between flow cytometry and florescence in situ hybridization (FISH) in 167 marrow specimens with MM. Gene expression profiling of CD56, CD117, CD52 and CD20 mRNA in plasma cells (PCs) from patients treated on Total Therapy 2 and Total Therapy 3 trials were also evaluated. Higher expression of CD56 and CD117 was associated with hyperdiploidy. High CD52 mRNA expression was associated with c-MAF and FGFR3 subgroups. Higher expression of CD56 mRNA, but lower Kit expression, were noted in association with FGFR3. In contrast, the c-MAF subgroup showed high Kit expression but lacked NCAM mRNA expression. CKS1B amplification showed positive correlation with CD52 (p = 0.0065) but negative correlation with CD20 (p = 0.0207). These findings indicate that phenotypic differences in MM are associated with distinct genetic subgroups, which potentially has important diagnostic and prognostic value.

A subset of CD20(+) MM patients without the t(11;14) are associated with poor prognosis and a link to aberrant expression of Wnt signaling.

To the editor Multiple myeloma (MM) is a hematologic malignancy, characterized by an excess of clonotypic plasma cells in the bone marrow [1]. Despite the development of new MM treatments that have significantly improved MM management (e.g., bortezomib, lenalidomide, pomalidomide, and thalidomide), many patients relapse and do not respond to current therapies, resulting in the death of the individual. Therefore, identification of a reliable and clinically useful biomarker is needed to develop an efficacious and nontoxic treatment options. Monoclonal antibody therapy targeting tumor cell surface receptors, signaling pathways or tumor-associated antigens has shown to be effective in improving overall cancer survival [2,3]. Currently, there are more than 15 monoclonal antibodies being investigated in preclinical and clinical MM trials [4]. Rituximab, one of the earliest antibodies evaluated in the treatment of lymphoma, works by targeting the B cellspecific antigen CD20 (http://www.gene.com/gene/products/ information/pdf/rituxan-prescribing.pdf). However, only minimal response to Rituximab therapy was achieved in MM patients [5]. In this study, we analyzed CD20 expression using a cohort of large clinical samples derived from newly diagnosed MM patients. Our findings can explain why the efficacy of Rituximab in the treatment of MMwas disappointing and demonstrate that CD20 can be used as not only a prognostic marker but also as a drug target, which may lead to more personalized treatment options for MM patients. +k;plasma-cell (NPC), 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 newly diagnosedMM patients, and 90 relapsedMM patients (RL-MM) were analyzed. Comparison of CD20 transcript expression among these groups revealed median signals from NPC, MGUS, MM, and RL-MM as 1681, 3210, 1990, and 1884, respectively (Figure 1A). Clearly, CD20 expression was increased in MGUS (45%), MM (18%), and RL-MM (12%) compared with NPC. Furthermore, the samples from the 351 MM patients were divided into three subgroups on the basis of increased expression of cyclin D1 (CCND1) compared with NPC CCND1 expression: low-CCND1 (Probe set: 208711_s_at, signal 0–600), medium-CCND1 (signal 601–5 000), and spike-CCND1 (signal>5 000). Previous report suggested that MM patients with spike-CCDN1 expression implies a t(11;14) [6]. We further examined the distribution of CD20 in MM patients