Abstract
MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the ...transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P<0.05, fold-change >1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P<0.05) in cases with high-levels of MYC. The MYC-mediated repression of miRNA levels was also observed (P<0.05) in B cells from transgenic MYC over-expressing mice (MYC controlled by an Ig-alpha enhancer) in comparison to wild-type animals (samples obtained from young animals before occurrence of any malignancy). We have further shown that the levels of this miRNA affect B cell proliferation in vitro, and its low-levels associate with percentage of Ki67 positive cells in FL samples (P<0.005). Moreover, low levels of tFL-associated miRNA were present in FL cases with a shorter overall survival (P<0.01), and its expression directly affected BCR signalling (calcium flux assay after anti-IgM). We have shown that the expression of this miRNA is not only down-modulated by high-level MYC expression, but also by B cell adhesion to stromal cells in co-culture in vitro (HS-5 stromal cells). This suggests that its normal physiological function might be related to regulation of B cell functions in the context of immune niches, and this might play a role in FL progression and transformation. It remains to be elucidated what other molecular mechanisms ensure low-level expression of the studied miRNA in cases that do not harbor MYC over-expression, and what pool of target mRNAs is regulated by this miRNA in FL cells.This work was supported by: the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. contact: marek.mraz@email.cz
Citation Format: Katerina Musilova, Gabriela Pavlasova, Vaclav Seda, Eva Vojackova, Katerina Cerna, Veronika Svobodova, Robert Pytlik, Vit Prochazka, Zuzana Prouzova, Sarka Pospisilova, Lenka Zlamalikova, Heidi Mocikova, Lenka Kruzova, Marie Jarosova, Andrew Evans, Clive Zent, Leos Kren, Marek Trneny, Jiri Mayer, Andrea Janikova, Marek Mraz. Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2017-1479
Abstract 3883
MicroRNAs (miRNAs) are known to be involved in the pathogenesis of chronic lymphocytic leukemia (CLL) and affect its clinical course (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012). ...Moreover, we and others have shown that several miRNAs are down-regulated in the aggressive CLL subtype harboring p53 aberration (Mraz et al. Leukemia, 2009). The role of miRNAs in primary or acquired resistance to therapy in CLL, however, is poorly understood.
In this study, we screened for miRNAs that are induced by fludarabine-mediated apoptosis in vitro, and we suggested that differences in the expression of one of the identified miRNAs (miR-34a) can be used to distinguish patients with impairment of the p53-apoptotic pathway.
Ten primary CLL B-cell samples (purity>95% of CD5+19+ cells) were treated in vitro with fludarabine dose (IC50 dose of 3.5 ug/mL, 48hrs), and the expression of 750 miRNAs was subsequently profiled (TaqMan miRNA Cards, ABI). In comparison with untreated control samples, 15 miRNAs were induced by fludarabine (fold change>1.5, SAM FDR<0.05). The most prominently up-regulated miRNA was miR-34a (fold change 3.7, P=0.003), which is a known p53 down-stream target (He et al. Nature, 2007). We then compared miR-34a up-regulation post fludarabine treatment to the decrease in cell viability (wt-p53 samples, N=20). This revealed that miR-34a induction was significantly higher in CLL samples more sensitive to fludarabine and suggested its role in the apoptotic effects of fludarabine in B-cells. Moreover, the up-regulation of miR-34a was also observed in vivo in samples obtained from fifty FCR-treated CLL patients (fold change 2.2, P<0.0001, analyzed at day 0 and 3 of FCR). These data encouraged us to develop an assay for absolute quantification of miR-34a which would allow determining the copy numbers of miR-34a, defining precise cut-offs, and comparing miR-34a levels during the course of the disease in one patient. We designed synthetic RNA oligos that were used to construct standard curves for both miR-34a and a normalization gene (RNU48). Using this assay, we profiled the expression of miR-34a in a cohort of CLL patients (N=200) to define a cut-off value that would discriminate therapy resistant cases. The distribution of miR-34a expression in the cohort ranged from 1 to 81820 molecules (per 10e6 copies of RNU48). Significantly, miR-34a levels below 2500 copies (N=47) were correlated with shorter overall survival (9.6 years vs. not-reached, HR 2.2 CI 1.1–4.5, P=0.03). Subsequently, the expression of miR-34a was compared in samples stratified by known prognostic markers: chromosomal aberrations (del17p13, del11q23, tris.12, and del13q14), IgHV status, expression of CD38, CD49d, age, gender, and Rai stage. The lower miR-34a levels were only associated with the deletion of 17p13 locus that includes the p53 gene (N=18, fold change −3.4, P=0.003). Remarkably, CLL samples with sole p53 mutation not accompanied by p53 deletion (N=13) also expressed low levels of miR-34a compared to wt-p53 (P=0.005, fold change −2.7). Most notably, 77% (10/13) of these samples had miR-34a levels below the cut-off of 2500 copies. We further validated our observations and assay by analyzing miR-34a expression in paired samples from 12 CLL cases that acquired p53 aberration during the course of the disease. This emphasized that miR-34a expression decreased in all cases after occurrence of p53 mutation (P=0.0008, fold change −6.1). Additionally, the effect of miR-34a up-regulation on therapy response is currently being investigated in a cohort of FCR-treated patients (N=50).
Our data provide complex evidence for the use of miR-34a as a marker of fludarabine-resistant disease. MicroRNA-34a quantification can identify p53 mutated cases that would not be recognized by FISH (mutation not accompanied by del17p13), and miR-34a down-regulation can be used as a sensor for acquisition of p53 abnormality during the course of the disease. This can be accomplished without treatment of cells with gamma-irradiation, which was previously used to identify functional impairment of the p53-pathway (Pettitt et al. Blood, 2001; Mous et al. Leukemia, 2009; Lin et al. CCR, 2012). The described assay for absolute quantification of miR-34a encourages further inter-laboratory validation.
IGA MZCR NT11218–6/2010, CZ.1.07/2.3.00/20.0045, CZ.1.05/1.1.00/02.0068
No relevant conflicts of interest to declare.
Abstract
The biology of B cell Non-Hodgkin lymphomas (NHLs) is largely influenced by (de)regulation of B cell receptor signaling (BCR sig.) and DNA damage response pathway (DDR). We and others have ...shown that in NHLs, miR-34a is involved in DDR, and miR-150/miR-155 are involved in BCR sig. (Mraz et al., 2009, 2014; Cui et al., 2014). To identify miRNAs involved in DDR and BCR sig. we used NGS technology (HiSeq) in chronic lymphocytic leukemia (CLL) B cells treated with BCR inhibitor or DNA damaging drugs. To investigate the DDR-related miRNAs, primary CLL cells were treated with fludarabine in vitro and paired samples (before/after treatment, n = 20) were analyzed for miRNAs’ profile. This identified 6 differentially expressed miRNAs (FDR<0.1). To investigate these miRNAs in DDR in vivo we profiled the expression of 3 out of the 6 miRNAs (miR-34a, miR-1246, miR-1248) in samples obtained from 50 CLL patients before and during the therapy containing fludarabine (FCR regiment). This confirmed up-regulation of all these miRNAs (fold change>1.5, P<0.05); according to our knowledge this is the first analysis of miRNA profiles during therapy administration in NHLs. Importantly, the miR-34a level was a significant predictor (p<0.05) of patients’ response to FCR therapy (complete response vs. others) and the progression free survival (19.9 vs. 26.4 mo.; HR: 2.29). A similar trend was observed for miR-1246, however, this was not statistically significant (P = 0.11). Additionally, low miR-34a is an independent predictor (in a multivariate analysis) of a shorter overall survival (16.7 mo. vs. not reached; P = 0.0002; HR: 3.30). This suggests that CLL cells with low levels of miR-34a fail to down-modulate genes that are crucial for DDR. Several pro-survival genes targeted by miR-34a were recently identified in various cell types (BIRC3, BCL2, FOXP1, YY1, Survivin). Some of these proteins were down-modulated in CLL B cells that up-regulate miR-34a during DDR or we have transfected with synthetic miR-34a, but not in cells that fail to induce miR-34a. Surprisingly, miR-34a, miR-1246 and miR-1248 share numerous validated and predicted targets with miR-150, a known negative regulator of BCR sig. (Mraz et al., 2014). This suggests possible convergence in the mechanism of action of DNA damaging drugs and BCR inhibitors recently approved for treatment of NHLs (such as ibrutinib). To compare the effects of FCR administration with the administration of ibrutinib, we analyzed miRNA and gene expression (HiSeq) in samples from ibrutinib treated CLL patients (n = 9) before and during the therapy. The convergence of pathways targeted by DDR and BCR inhibition through changes in miRNAs’ expression are currently being interrogated.
Supported by: SoMoPro II-no. 4SGA8684; NGS-PTL (306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0830/2013; MH CZ-DRO (FNBr, 65269705), CZ.1.05/1.1.00/02.0068, CZ.1.07/2.3.00/30.0009.
Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Vaclav Seda, Gabriela Pavlasova, Michal Jez, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. MicroRNA involvement in DNA damage response and BCR signaling in malignant B cells. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2015-3084
Abstract The technology of array comparative genomic hybridization (array-CGH/aCGH) enabled the identification of novel genomic aberrations in chronic lymphocytic leukemia (CLL) including the ...monoallelic and biallelic deletions affecting 22q11 locus. In contrast to previous publications, we hypothesized that the described 22q11 deletions are a consequence of the rearrangement of immunoglobulin lambda light chain locus (IGL) segments surrounding several protein-coding genes located in this region. Indeed, using array-CGH and PCR analysis we show that all deletions ( n = 7) affecting the 22q11 locus in our cohort ( n = 40) are based on the physiological mechanism of IGL rearrangement. This demonstrates that this loss of genetic material is likely not pathogenic and in fact is merely a marker of IGL rearrangement.
Abstract
We and others have shown that expression of miRNAs influences the biology of B cell malignancies (Calin et al., 2005; Mraz et al., 2009, 2012, 2013). The aim of this study was to identify ...miRNAs involved in the apoptotic response of malignant B cells. Purified primary B cells of chronic lymphocytic leukemia (CLL) patients (pts.) were treated in vitro with fludarabine (F) (LC50 dose of 3.5 μg/ml; 48 h). Five paired (n=10) samples (with and without F) were analyzed using 2 NGS platforms-SOLiD (ABI) and HiSeq (Illumina). The obtained reads were mapped to miRBase using 3 tools (CLC Genomic Workbench, SHRiMP2, miRanalyzer) and data analyzed by a pair-wise comparison with edgeR and baySeq packages (Bioconductor 2.13). The overlap of these 3+2 bioinformatic approaches identified 6 miRNAs significantly changed with DNA damage. RNA-Seq validation on 5 additional paired samples (n=10) confirmed the changed expression of all 6 previously identified miRNAs (3 down-, 3 up-regulated). The most constantly up-regulated was miR-34a, which was previously shown to be regulated by p53 (He et al., 2005; Mraz et al., 2009). To test the importance of miR-34a in vivo, we collected samples from CLL pts. (n=51) treated with F, cyclophosphamide and rituximab (FCR) regimen. miR-34a was induced in all samples after F administration (day 2, p<0.001). Surprisingly, the lower basal and induced levels of miR-34a correlated with significantly (p<0.05) shorter time to treatment failure, suggesting its strong prognostic potential. We further determined the expression of miR-34a in a large cohort of CLL pts. (n=158) using an in-house designed assay for its copy-number quantification. We defined a cut-point (number of miR-34a copies) that segregates pts. with extremely unfavorable prognosis (overall survival OS 1.37 yrs. vs. not reached; p=0.0001; HR=3.89; CI=2.05-7.39) and this was independent of routinely used prognostic markers (FISH, IgHV, age, sex) in a multivariate analysis. We have previously described that low levels of miR-34a associate with TP53 abnormalities, so we limited the analysis to wt-TP53 samples (n=116). In this multivariate analysis miR-34a was the strongest predictor of OS (1.29 yrs. vs. not reached; p=0.002; HR=9.82; CI=2.30-42.05). The molecular pathways affected by miR-34a levels in B cells are largely unknown. However, miR-34a shares 51 predicted target mRNAs (evolutionary conserved) with at least 1 other F induced miRNA, suggesting that they might cooperate in the regulation of these genes. The pathways regulated by these miRNAs are currently being investigated using integrated analysis of miRNA and transcriptome profiling of CLL samples (n=100).
Supported by: NGS-PTL (FP7-HEALTH-2012-INNOVATION-1, no. 306242); EHA Research Fellowship award; Grant Agency of Czech Rep.; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0723/2012; CZ.1.07/2.3.00/30.0009, co-financed from EU and Czech Rep.
Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2014-5198
Abstract 3874
It is known that the biology of CLL and other B-cell malignancies is driven by processes dependent on immunoglobulin structure and BCR-signaling. Recently, our group and others have ...described the importance of miRNAs in CLL and their involvement in BCR-signaling, IgG production and V(D)J recombination. Considering the important functions of miRNAs it is remarkable that the human locus for immunoglobulin lambda light chain (IgL) contains a miR gene. The miR-650 gene is localized in exon 1 of IgL variable subgene (1st exon of V2 family members). The aim of this study was to reveal the regulation and expression of miR-650 in CLL and its relation to disease prognosis.
CLL samples were separated by RosetteSep Human B Cell Enrichment Cocktail (obtained purity ≥95% of CD5+19+ cells). The expression of light chain surface immunoglobulin chain (lambda vs kappa) was determined by flow cytometry. The utilized IgL variable (V) segment was determined using BIOMED-2 protocol and sequencing. To study the relation of miR-650 expression and IgL rearrangement the surface expression of Ig light chain and the utilized V segment was determined in a CLL cohort containing 47 patients (λ n=27, κ n=19, λ+κ n=1). The gene expression was analyzed by TaqMan Assays (ABI) for mature miR-650 and protein-coding genes: CDK1, EBF3 and ING4. The western-blot for miR-650 targets was performed after transfection (48hrs/96hrs) of B-cell lines (NALM-6, MEC-1) with a short RNA mimicking miR-650 (Dharmacon).
The analyses of miR-650 expression revealed that cells utilizing V2-8, V2-5, V2-14, V2-23 subgenes for IgL (n=14) had ∼10 fold higher expression of miR-650 (p<0.005) when compared to samples utilizing different V lambda family (n=13) or expressing kappa Ig (n=20). This suggests a unique mechanism for coordinate expression of miR-650 and immunoglobulin light chain (for IgL utilizing the V2 family members). This observation is partially surprising because miR-650 was originally identified in colorectal and breast cells and it was believed to be regulated independently of immunoglobulin genes. Our data demonstrate that miR-650 expression is likely regulated at least partially by immunoglobulin light chain promoter.
We next studied the possible targets for miR-650 in B-cells. Recently, two targets were identified in solid tumors - ING4 (Inhibitor of Growth 4) and CDK1 (cyclin dependent kinase 1) (Zhang, 2010; Chien, 2010). It has been demonstrated that miR-650 is involved in the p16INK4-mediated pathway and directly regulates the CDK1. This publication suggested that up-regulation of miR-650 leads to inhibition of cell cycle progression. Moreover, the putative targets predicted by software tools (TargetScan, miRanda) include genes important for B-cell biology like EBF3 (early B-cell factor3), CLLU1, Bcl2 and cyclin D1. We therefore studied correlation between miR-650 expression and the expression of mRNAs for CDK1, ING4 and EBF3 (a predicted target with the highest score). The expression of miR-650 was not significantly associated with the expression of any of these genes on mRNA level. The lack of available material did not allow us to study the expression of CDK1, ING4, EBF3 protein levels in the original cohort. However, the transfection of B-cells with short RNA mimicking miR-650 led to down-regulation of protein levels of CDK1 and EBF3. This confirms the relevance of CDK1 as a target in B-cells and identifies a new target - EBF3, which is known to be important for B-cells development. Moreover, the expression of miR-650 was associated with overall survival (OS) and treatment free survival (TFS) in CLL (n=82). In this analysis patients were divided in two groups (based on the median of miR-650 expression). The higher expression of miR-650 was associated with statistically significant (p<0.05) longer OS (not-reached vs. 161 months) and TFS (60 vs. 34 months). This is in line with the observation that miR-650 inhibits CDK1 and cell cycle progression.
In conclusion, we have described a mechanism regulating miR-650 expression, identified its relevant targets in B-cells and demonstrated the association of miR-650 expression with CLL prognosis.
Supported by IGA MZCR NT11218-6/2010, MSMTMSM0021622430, NS10439-3/2009, FR-TI2/254
Mayer:Roche: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Astellas: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Novartis: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Fresenius Medical Care: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Pfizer: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Genzyme: Consultancy, Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; GSK: Honoraria, Institutional/personal grants and travel/accommodation expenses, Speakers Bureau; Amgen:.
