Distinct Innate Immunity Pathways to Activation and Tolerance in Subgroups of Chronic Lymphocytic Leukemia with Distinct Immunoglobulin Receptors

Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway–associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulation of TLRs and NOD2 in 67 CLL cases assigned to different subgroups on the basis of immunoglobulin heavy variable (IGHV ) gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of costimulatory molecules and/or apoptosis were observed in mutated versus unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that “subset-biased” innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

Relevance of stereotyped B-cell receptors in the context of the molecular, cytogenetic and clinical features of chronic lymphocytic leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.     

Prognostic impact of epigenetic classification in chronic lymphocytic leukemia: The case of subset #2

Based on the methylation status of 5 single CpG sites, a novel epigenetic classification of chronic lymphocytic leukemia (CLL) was recently proposed, classifying CLL patients into 3 clinico-biological subgroups with different outcome, termed memory like CLL (m-CLL), naïve like CLL (n-CLL), and a third intermediate CLL subgroup (i-CLL). While m-CLL and n-CLL patients at large corresponded to patients carrying mutated and unmutated IGHV genes, respectively, limited information exists regarding the less defined i-CLL group. Using pyrosequencing, we investigated the prognostic impact of the proposed 5 CpG signature in a well-characterized CLL cohort (135 cases), including IGHV-mutated and unmutated patients as well as clinically aggressive stereotyped subset #2 patients. Overall, we confirmed the signature's association with established prognostic markers. Moreover, in the presence of the IGHV mutational status, the epigenetic signature remained independently associated with both time-to-first-treatment and overall survival in multivariate analyses. As a prime finding, we observed that subset #2 patients were predominantly classified as i-CLL, probably reflecting their borderline IGHV mutational status (97–99% germline identity), though having a similarly poor prognosis as n-CLL patients. In summary, we validated the epigenetic classifier as an independent factor in CLL prognostication and provide further evidence that subset #2 is a member of the i-CLL group, hence supporting the existence of a third, intermediate epigenetic subgroup.     

MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+) and IgV(H) unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.     

Mutation pattern of paired immunoglobulin heavy and light variable domains in chronic lymphocytic leukemia B cells

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.     

Cooperative miRNA-dependent PTEN regulation drives resistance to BTK inhibition in B-cell lymphoid malignancies

Aberrant microRNA (miR) expression plays an important role in pathogenesis of different types of cancers, including B-cell lymphoid malignancies and in the development of chemo-sensitivity or -resistance in chronic lymphocytic leukemia (CLL) as well as diffuse large B-cell lymphoma (DLBCL). Ibrutinib is a first-in class, oral, covalent Bruton’s tyrosine kinase (BTK) inhibitor (BTKi) that has shown impressive clinical activity, yet many ibrutinib-treated patients relapse or develop resistance over time. We have reported that acquired resistance to ibrutinib is associated with downregulation of tumor suppressor protein PTEN and activation of the PI3K/AKT pathway. Yet how PTEN mediates chemoresistance in B-cell malignancies is not clear. We now show that the BTKi ibrutinib and a second-generation compound, acalabrutinib downregulate miRNAs located in the 14q32 miRNA cluster region, including miR-494, miR-495, and miR-543. BTKi-resistant CLL and DLBCL cells had striking overexpression of miR-494, miR-495, miR-543, and reduced PTEN expression, indicating further regulation of the PI3K/AKT/mTOR pathway in acquired BTKi resistance. Additionally, unlike ibrutinib-sensitive CLL patient samples, those with resistance to ibrutinib treatment, demonstrated upregulation of 14q32 cluster miRNAs, including miR-494, miR-495, and miR-543 and decreased pten mRNA expression. Luciferase reporter gene assay showed that miR-494 directly targeted and suppressed PTEN expression by recognizing two conserved binding sites in the PTEN 3′-UTR, and subsequently activated AKT^Ser473. Importantly, overexpression of a miR-494 mimic abrogated both PTEN mRNA and protein levels, further indicating regulation of apoptosis by PTEN/AKT/mTOR. Conversely, overexpression of a miR-494 inhibitor in BTKi-resistant cells restored PTEN mRNA and protein levels, thereby sensitizing cells to BTKi-induced apoptosis. Inhibition of miR-494 and miR-495 sensitized cells by cooperative targeting of pten, with additional miRNAs in the 14q32 cluster that target pten able to contribute to its regulation. Therefore, targeting 14q32 cluster miRNAs may have therapeutic value in acquired BTK-resistant patients via regulation of the PTEN/AKT/mTOR signaling axis.Subject terms: Cancer, Chronic lymphocytic leukaemia     

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background

Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBL^hi) or without (MBL^lo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.

Methodology/Principal Findings

For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBL^lo clones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBL^hi and advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)⁺ clonal B-cells.

Conclusions/Significance

These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBL^lo clones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution.     

MicroRNA-26a negatively regulates toll-like receptor 3 expression of rat macrophages and ameliorates pristane induced arthritis in rats

Introduction

Abnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. This study was performed to determine the relationship between miR-26a and TLR3 in rat macrophages and to observe effects of miR-26a mimic on pristane induced arthritis (PIA) in rats.

Methods

Dual luciferase reporter assay was used to validate the direct interaction between miR-26a (a candidate miRNA to target tlr3 mRNA) and tlr3 3′UTR. MiR-26a regulation on TLR3 gene expression was determined using RT-qPCR and Western blotting after miR-26a mimics and inhibitors were transfected into rat macrophage line NR8383 cells. Poly I:C (TLR3 ligand) was used to trigger TLR3 activation, and mRNA expression of its downstream cytokines interferon (ifn)-β and tumor necrosis factor (tnf)-α was accordingly detected to determine the regulation of TLR3 signaling. Expressions of TLR3 and miR-26a were detected during rat bone marrow derived macrophage (BMDM) induction, in pristane stimulated NR8383 cells and spleens from methotrexate (MTX) treated PIA rats. A miR-26a mimic was administrated intraperitoneally to PIA rats, and arthritis severity was evaluated by macroscopic or microscopic observations.

Results

Direct target relationship between miR-26a and tlr3 mRNA in rats was confirmed. Modifications of miR-26a function by transfection of miR-26a mimics and inhibitors exhibited corresponding repression and augmentation of TLR3 and its signaling downstream cytokine expressions in NR8383 cells. The alteration of miR-26a expression was negatively related with TLR3 expression during BMDM induction, in pristane-primed NR8383 cells and PIA rat spleens. Moreover, both abnormal expressions were rescued in MTX treated arthritis rat spleens. The miR-26a mimic treatment displayed the depression of TLR3 expression and ameliorated the disease severity in the rats with pristane induced arthritis.

Conclusions

MiR-26a negatively regulates TLR3 signaling via targeting of TLR3 itself in rat macrophages, and this finding provides a novel insight into abnormal TLR3 overexpression during experimental arthritis.     

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct,an Immunodominant Oxidation-Specific Epitope

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.     

Toll-like receptor 2 activation implicated in oral squamous cell carcinoma development

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity; however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2 was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived from bacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signal transduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly, ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of caspase recruitment domain–containing protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. These findings suggest that the activation of TLR2 by bacterial components can enhance the progression of OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of the miR-146a pathway.     

Exosome-encapsulated miR-6089 regulates inflammatory response via targeting TLR4

Exosome-encapsulated microRNAs (miRNAs) have been identified as potential biomarkers in autoimmune diseases. However, little is known about the role of exosome-delivered miRNAs in rheumatoid arthritis (RA). In this study, we investigated the profile of specific exosomal miRNAs by microarray analysis of serum exosomes from three patients with RA and three healthy controls. Quantitative real-time PCR (qRT-PCR) was performed to validate the aberrantly expressed exosomal miRNAs. A total of 20 exosome-encapsulated miRNAs were identified to be differently expressed in the serum of patients with RA compared with controls. Interestingly, we found that exosome-encapsulated miR-6089 was significantly decreased after validation by qRT-PCR in serum exosomes from 76 patients with RA and 20 controls. Besides, miR-6089 could inhibit lipopolysaccharide (LPS)-induced cell proliferation and activation of macrophage-like THP-1 cells. TLR4 was a direct target for miR-6089. MiR-6089 regulated the generation of IL-6, IL-29, and TNF-α by targetedly controlling TLR4 signaling. In conclusion, exosome-encapsulated miR-6089 regulates LPS/TLR4-mediated inflammatory response, which may serve as a novel, promising biomarker in RA.     

MicroRNA expression and clinical outcome of small cell lung cancer

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.     

MicroRNA-7–regulated TLR9 signaling–enhanced growth and metastatic potential of human lung cancer cells by altering the phosphoinositide-3-kinase,regulatory subunit 3/Akt pathway

Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology.