Letter to the Editor | Published:

A microRNA signature specific for hairy cell leukemia and associated with modulation of the MAPK–JNK pathways

Leukemia volume 26, pages 25642567 (2012) | Download Citation

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder characterized by marked splenomegaly, pancytopenia and proliferation of tumor cells with hairy appearance that infiltrate the spleen, liver and bone marrow and circulate at low percentage in the peripheral blood.1 HCL displays a unique immunophenotype (ANXA1+, CD103+, CD25+ and CD11c+) that is distinct from that of other B-cell lymphomas.1 HCL is highly sensitive to purine analogues that induce a response in about 90% of patients.1

During the past decade, significant progress has been made in the understanding of the HCL pathogenesis.1 Gene-expression profile analysis identified a unique molecular signature, which suggested a derivation from memory B cells, provided novel diagnostic markers and explained some of the peculiar features of leukemic hairy cells.2 In particular, the expression of genes controlling cell adhesion and response to chemokines appeared to be significantly altered in HCL, supporting the aberrant adhesion and homing properties of HCL cells.2 No recurrent chromosomal alterations have been identified in HCL, while studies based on genome-wide single-nucleotide polymorphism analysis confirmed a remarkably stable genome.1, 3 More recently, using a whole-exome sequencing approach, the BRAF V600E gene mutation was identified as the first genetic hallmark of HCL.4 The presence of BRAF-activating mutations in virtually all HCL cases4, 5 strongly suggests that the RAF-MEK-ERK pathway may have a critical role in the HCL pathogenesis.

A novel layer of biological complexity has been added by the discovery of microRNAs (miRNAs) and their implication in cancer. In B cells, miRNAs are expressed in a stage- or transformation-specific fashion, suggesting that they might have developmental and pathological roles.6 Although miRNA-expression signatures have been derived for several hematological malignancies, the contribution of miRNAs to HCL has not yet been analyzed. In order to investigate the potential role of miRNAs in HCL pathogenesis and the possibility of using miRNA-expression signatures for the differential diagnosis of HCL, miRNA-expression profiling was performed on peripheral blood-derived CD19+ B cells from eight HCL patients (all harboring the BRAF V600E gene mutation4), five patients with splenic lymphoma with villous lymphocytes (SLVLs; all devoid of the BRAF V600E gene mutation) and nine patients with B-cell chronic lymphocytic leukemia (B-CLL; of which five could be tested and turned out to be negative for the BRAF V600E gene mutation) using the Human miRNA microarray kit v.1.0 (Agilent Technologies, Santa Clara, CA, USA). For comparison, six each naïve, germinal center and memory B-cell samples from healthy donors were also included in the study. Unsupervised hierarchical clustering, based on the average linkage method and Pearson’s correlation,7 was used to interrogate the miRNA-expression profiles for their ability of discriminating HCL, SLVL and B-CLL. The results showed that HCL displayed a unique and homogenous phenotype clearly distinct from that of the other B-cell malignancies (Figure 1a). Similarly, B-CLL tended to cluster together, whereas SLVL did not form a uniform cluster likely because of their higher heterogeneity (Figure 1a).

Figure 1
Figure 1

HCL display a homogenous miRNA-expression profile. (a) Hierarchical clustering based on miRNA-expression profiles of HCL, SLVL and B-CLL. The analysis is based on the subset of miRNAs displaying at least a two-fold average change in expression levels from the mean across the whole panel. (b) Identification of a HCL-specific miRNA signature by supervised analysis. The Z-score is displayed for each miRNA.

In order to identify a HCL-specific miRNA signature, HCLs were compared with SLVLs, B-CLLs and normal mature B cells at different stages of differentiation including naïve, memory and germinal-center B cells. This supervised analysis was performed using the Genes@Work software platform8 and identified six miRNAs, which were expressed at significantly higher level in HCL compared with normal and other malignant B cells (Figure 1b). The HCL-specific signature included the miR-221/miR-222 family, miR-22, miR-24, miR-27a and let-7b.

MiRNAs exert their function by negatively modulating the translation of their targets, and several genes have been already demonstrated to be directly targeted by the miRNAs identified here as deregulated in HCL. Interestingly, CDKN1B (p27/Kip1) represents a fully characterized direct target of miR-221/miR-222.9 Low levels of CDKN1B protein expression in HCL have been previously reported and could not be explained by genetics, decreased transcription or increased ubiquitin-mediated degradation.10 Therefore, it has been suggested that low CDKN1B protein levels in HCL may be due to unknown mechanisms of posttranscriptional regulation.10 Our data strongly suggest that miR-221/miR-222 overexpression in HCL represents at least one mechanism, leading to low CDKN1B protein expression in HCL.

MiR-24 has been demonstrated to directly target CDKN2A protein (p16),11 a tumor suppressor, largely affected by gene deletions in human cancer, but not in HCL.3 Overexpression of miR-24 in HCL may lead to a negative modulation on CDKN2A protein in the absence of lesions affecting the gene locus. The CDKN1A (p21) protein has been recently reported to be regulated by miR-22, whose overexpression in HCL could contribute to make HCL cells more resistant to apoptosis.12

To gain further insights on the potential role of miRNAs in HCL pathogenesis, we applied five target prediction algorithms (MiRBase Targets, TargetScan, PicTar, RNA22 and PITA)13 to identify the candidate targets of the six-miRNA HCL-specific signature. Targets commonly predicted by at least four of the prediction methods and expressed in B cells were further investigated. Overall, 257 genes were predicted to be targeted by at least one of the HCL-overexpressed miRNAs. The pathway-enrichment analysis was performed on the 257 predicted targets using the Database for Annotation, Visualization and Integrated Discovery.14 The mitogen-activated protein kinase (MAPK) pathway appeared as the most significantly enriched in predicted targets of HCL-overexpressed miRNAs, suggesting that miRNA-deregulated expression in HCL may modulate MAPK signaling (Figure 2). Interestingly, most of the predicted targets belonging to the MAPK signaling pathway were involved in the activation of p38 MAPK and Jun N-terminal kinase (JNK). This pathway has been shown to make HCL cells susceptible to apoptosis, an effect likely triggered by the interaction of HCL cells with the microenviroment, in particular with the vitronectin-positive cells in the splenic red pulp.15 Of note, miRNA-mediated repression was not predicted for proteins in the RAF-MEK-ERK pathway, consistent with a lack of negative modulation of this pathway, which is constitutively active in HCL cells because of BRAF somatic mutation.4 The balance between ERK and JNK/p38 MAPK may have a critical role in HCL, as suggested by the observation that active JNK/p38 MAPK makes HCL cells susceptible to apoptosis, whereas they are effectively rescued by ERK activation.15 Therefore, negative modulation of the pro-apoptotic JNK/p38 MAPK signaling pathways caused by overexpression of miRNAs may contribute to make HCL cells more resistant to apoptosis. This mechanism may act in concert with the constitutive activation of the RAF-MEK-ERK pathway triggered by the BRAF V600E mutant.

Figure 2
Figure 2

MAPK signaling pathway is significantly enriched for targets predicted to be affected by the HCL miRNA signature. A total of 257 targets were predicted by at least four prediction algorithms to be affected by the HCL miRNA signature. The pathway-enrichment analysis was performed on the 257 predicted targets and significant enrichment (upon Bonferroni correction) was detected only for the MAPK signaling pathway. The red label identifies predicted targets. The color reproduction of this figure is available at the Leukemia journal online.

Taken together, the data herein identify a HCL-specific miRNA signature with implications for the pathogenesis and clinical management of HCL. In fact, these results reinforce the notion that the MAPK pathway has a critical role in the development of HCL. This in turn suggests a druggable target because many available compounds can modulate the altered MAPK–JNK pathway typical of leukemic hairy cells.

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Acknowledgements

BF was supported by an investigator grant from the Associazione Italiana Ricerca sul Cancro and ET was supported by a fellowship (2008/14) from the European Hematology Association. We thank Roberta Pacini and Gianluca Schiavoni for their technical assistance.

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Author notes

    • B Falini
    •  & K Basso

    These two authors contributed equally to this work.

Affiliations

  1. Institute for Cancer Genetics and H. Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA

    • Y Kitagawa
    • , M Brahmachary
    • , R Dalla-Favera
    •  & K Basso
  2. Institute for Integrated Medical Sciences, Tokyo Women’s Medical University, Tokyo, Japan

    • Y Kitagawa
  3. Joint Centers for Systems Biology, Columbia University, New York, NY, USA

    • M Brahmachary
  4. Institute of Hematology, University of Perugia, Perugia, Italy

    • E Tiacci
    •  & B Falini
  5. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    • R Dalla-Favera
    •  & K Basso
  6. Department of Genetics and Development, Columbia University, New York, NY, USA

    • R Dalla-Favera
  7. Department of Microbiology and Immunology, Columbia University, New York, NY, USA

    • R Dalla-Favera

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Competing interests

BF and ET applied for a patent on the clinical use of the BRAF V600E mutant in HCL. The remaining authors declare no conflict of interest.

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Correspondence to B Falini.

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DOI

https://doi.org/10.1038/leu.2012.149

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