Clinical correlation of miR-155 and TP53 expression with survival in the lung adenocarcinoma - TCGA dataset when distinguishing between TP53 wild-type and TP53 mutated samples. (A-B) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in samples that express wild-typeTP53 or harbor TP53 mutations that do not affect TP53 function (A), and in samples expressing mutated TP53 that affects TP53 function (B). (C-D) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 and low levels of TP53 vs. low levels of miR-155 and high levels of TP53 in samples that express wild-type TP53 or harbor TP53 mutations that do not affect TP53 function (C), and in samples expressing mutated TP53 that affects TP53 function (D).

Dose-response curves for MEC1 and MEC2 cells treated with fludarabine.

Supplementary Table S1 Clinical characteristics of two chronic lymphocytic leukemia patient datasets; Supplementary Table S2 Clinical characteristics of two lung cancer patient datasets; Supplementary Table S3 Clinical characteristics of the ALL dataset; Supplementary Table S4 Integrated function and pathway analysis on 248 experimentally validated targets of miR-155; Supplementary Table S5 Univariate and multivariate analyses of survival with patient characteristics and miR-155 and TP53 expression as categorical and continuous variables in different patient cohorts; Supplementary Table S6 Estimate of Cox model and multivariate Cox model, as well as the HR estimated based on the model for miR-155 high and TP53 low vs. miR-155 low and TP53 high.

Clinical correlation of miR-155 expression with survival in leukemia. Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in two CLL cohorts, CLL - NEJM (A) and CLL - Italy (B), and in one ALL cohort, ALL - MDACC (C).

Supplementary Table 1: Inclusion and exclusion criteria. Supplementary Table 2: Lenalidomide dose reduction steps. Supplementary Table 3: Overall and complete response rates according to pre-treatment characteristics. Supplementary Table 4: Causes of death.

In vivo orthotopic lung cancer model for the role of miR-155 in chemoresistance. (A) Injection and treatment schedule for CDDP (green arrows) and anti-miR negative control (NC) or anti-miR-155 liposomal nanoparticles (red stars) for four different treatment groups: mice that were injected with A549-LVEV cells and untreated (group 1), injected with A549-LVEV cells and treated with anti-miR-NC and CDDP (group 2), injected with A549-155LV cells and treated with anti-miR-NC and CDDP (group 3), and injected with A549-155LV cells and treated with anti-miR-155 and CDDP (group 4). (B) Representative pictures of dissected mice belonging to each of the treatment groups described in panel A of this Figure. Tumor nodules are marked by dotted white circles. (C-D) Graphs of the primary tumor size (C) and aggregate mass of nodules in mediastinum (D) of the four treatment groups mentioned above. (E) In situ hybridization for miR-155 for each of the four treatment groups mentioned above. (F) Immunohistochemical analysis for Ki-67 (proliferation) and CD31 (angiogenesis), as well as the TUNEL assay (apoptosis) and TP53 immunostaining for each of the four treatment groups mentioned above.

Supplementary Figure 1: Ofatumumab and lenalidomide treatment schema. Supplementary Figure 2: Absolute number of responses and response rates in evaluable patients over time. Supplementary Figure 3: FACS plots showing baseline NK and T cell function in HD and CR, PR and NR patients. Supplementary Figure 4: Plasma chemokine and cytokine modulation during ofatumumab and lenalidomide treatment. Supplementary Figure 5: Changes in circulating angiogenic factors during therapy with ofatumumab and lenalidomide.

The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

Small non-coding microRNAs (miRNAs) are involved in virtually all physiological processes and deregulated in many human diseases. They exert their gene silencing function through association with Argonaute (AGO) proteins in the RNA-induced silencing complex (RISC). AGO proteins have been found to be deregulated in human diseases, including cancer, and are thus analogous to miRNAs as attractive therapeutic targets. The best known and most successful miRNA therapeutic, miravirsen, which was successfully evaluated in phase 2 clinical trials for the treatment of hepatitis C virus (HCV) infection, is a specific miR-122 inhibitor with a dual function of blocking AGO2 inhibition, supporting the potential of AGO-based therapeutics for the treatment of human diseases. This chapter first discusses the miRNA biogenesis process and the Argonaute family of proteins (AGO). Next, the mechanisms of AGO-mediated gene silencing are explained. The final section describes miRNA-based therapeutics, where we focus on therapeutic strategies of targeting AGO proteins for the treatment of human diseases, with an emphasis on cancer.

Small non-coding microRNAs (miRNAs) are instrumental in physiological processes, such as proliferation, cell cycle, apoptosis, and differentiation, processes which are often disrupted in diseases like cancer. miR-155 is one of the best conserved and multifunctional miRNAs, which is mainly characterized by overexpression in multiple diseases including malignant tumors. Altered expression of miR-155 is found to be associated with various physiological and pathological processes, including hematopoietic lineage differentiation, immune response, inflammation, and tumorigenesis. Furthermore, miR-155 drives therapy resistance mechanisms in various tumor types. Therefore, miR-155-mediated signaling pathways became a potential target for the molecular treatment of cancer. In this review, we summarize the current findings of miR-155 in hematopoietic lineage differentiation, the immune response, inflammation, and cancer therapy resistance. Furthermore, we discuss the potential of miR-155-based therapeutic approaches for the treatment of cancer.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity.

MicroRNAs (miRNAs) are small noncoding RNAs that target specific mRNAs through interaction with complementary sequences usually found in the 3'-untranslated regions (UTRs) of target mRNAs. miRNAs have been shown to play a fundamental role in the management of chronic lymphocytic leukemia (CLL) by modulating gene expression patterns and cellular signaling pathways. In recent years, several studies have focused on the role of regulatory miRNAs in the pathogenesis of CLL. Aberrant expression of CLL-specific miRNAs has emerged as therapeutic and diagnostic biomarkers in patients with CLL. Here, we describe a method for the quantification of miRNAs in malignant B cells from the mononuclear cell compartment, isolated from peripheral blood. We focus on the isolation of human blood monocytes by Ficoll-Paque gradient centrifugation, total RNA extraction from human peripheral blood mononuclear cells, and quantitative reverse transcription (qRT)-PCR, which is useful for the measurement of miRNAs in monocytes isolated from blood samples.

Human cancers are characterized by a number of hallmarks, including sustained proliferative signaling, evasion of growth suppressors, activated invasion and metastasis, replicative immortality, angiogenesis, resistance to cell death, and evasion of immune destruction. As microRNAs (miRNAs) are deregulated in virtually all human cancers, they show involvement in each of the cancer hallmarks as well. In this chapter, we describe the involvement of miRNAs in cancer from a cancer hallmarks and targeted therapeutics point of view. As no miRNA-based cancer therapeutics are available to date, and the only clinical trial on miRNA-based cancer therapeutics (MRX34) was terminated prematurely due to serious adverse events, we are focusing on protein-coding miRNA targets for which targeted therapeutics in oncology are already approved by the FDA. For each of the cancer hallmarks, we selected major protein-coding players and describe the miRNAs that target them.

Mammalian cells can release different types of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies. Accumulating evidence suggests that EVs play a role in cell‐to‐cell communication within the tumor microenvironment. EVs’ components, such as proteins, noncoding RNAs [microRNAs (miRNAs), and long noncoding RNAs (lncRNAs)], messenger RNAs (mRNAs), DNA, and lipids, can mediate paracrine signaling in the tumor microenvironment. Recently, miRNAs encapsulated in secreted EVs have been identified in the extracellular space. Mature miRNAs that participate in intercellular communication are released from most cells, often within EVs, and disseminate through the extracellular fluid to reach remote target cells, including tumor cells, whose phenotypes they can influence by regulating mRNA and protein expression either as tumor suppressors or as oncogenes, depending on their targets. In this review, we discuss the roles of miRNAs in intercellular communication, the biological function of extracellular miRNAs, and their potential applications for diagnosis and therapeutics. We will give examples of miRNAs that behave as hormones.

Abstract Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease and has a highly variable clinical course with survival ranging from a couple of months to several decades. MicroRNAs (miRNAs), small non-coding RNAs that regulate transcription and translation of genes, have been found to be involved in CLL initiation, progression, and resistance to therapy. In addition, they can be used as prognostic biomarkers and as targets for novel therapies. In this review, we describe the association between miRNAs and the cytogenetic aberrations commonly found in CLL, as well as with other prognostic factors. We describe the presence of miRNAs as extracellular entities in the plasma and serum of CLL patients and discuss their role in resistance to therapy. Finally, we will explore the potential of targeted miRNA therapy for the treatment of CLL, with a special emphasis on MRX34, the first miRNA mimic that is currently being evaluated for clinical use.

The recurrent 9p24.1 aberrations in lymphoid malignancies potentially involving four cancer‐related and druggable genes (JAK2, CD274/PDL1, PDCD1LG2/PDL2, and KDM4C/JMJD2Cl) are incompletely characterized. To gain more insight into the anatomy of these abnormalities, at first we studied 9p24.1 alterations in 18 leukemia/lymphoma cases using cytogenetic and molecular techniques. The aberrations comprised structural (nine cases) and numerical (nine cases) alterations. The former lesions were heterogeneous but shared a common breakpoint region of 200 kb downstream of JAK2. The rearrangements predominantly targeted the PDL locus. We have identified five potential partner genes of PDL1/2: PHACTR4 (1p34), N4BP2 (4p14), EEF1A1 (6q13), JAK2 (9p24.1), and IGL (22q11). Interestingly, the cryptic JAK2‐PDL1 rearrangement was generated by a microdeletion spanning the 3′JAK2−5′PDL1 region. JAK2 was additionally involved in a cytogenetically cryptic IGH‐mediated t(9;14)(p24.1;q32) found in two patients. This rare but likely underestimated rearrangement highlights the essential role of JAK2 in B‐cell neoplasms. Cases with amplification of 9p24.1 were diagnosed as primary mediastinal B‐cell lymphoma (five cases) and T‐cell lymphoma (four cases). The smallest amplified 9p24.1 region was restricted to the JAK2‐PDL1/2‐RANBP6 interval. In the next step, we screened 200 cases of classical Hodgkin lymphoma by interphase FISH and identified PDL1/2 rearrangement (CIITA‐ and IGH‐negative) in four cases (2%), what is a novel finding. Forty (25%) cases revealed high level amplification of 9p24.1, including four cases with a selective amplification of PDL1/2. Altogether, the majority of 9p24.1 rearrangements occurring in lymphoid malignancies seem to target the programmed death‐1 ligands, what potentiates the therapeutic activity of PD‐1 blockade in these tumors. © 2016 Wiley Periodicals, Inc.