We analyzed the expression profile of 29 CLL patients who were treated by fludarabine, a pivotal drug in the modern treatment of CLL [25, 26]. To better elucidate the molecular mechanisms underlying the development of fludarabine resistance, we investigated the miRNA expression alterations of patients who received fludarabine as single agent, thus avoiding the potentially confounding elements that are inevitably present when fludarabine is used in combination with other drugs. Moreover, samples were obtained before and after first fludarabine treatment from the same patient, making it possible to directly evaluate the effect of the drug in vivo.
Our results revealed that many miRNAs are modulated by fludarabine treatment, as shown by the 37 miRNAs that are modulated following the treatment in both CR and NR patients, while 23 and 30 miRNAs were differentially modulated only in CRs or NRs, respectively, suggesting that these two cohorts of patients show signs of differences in the execution of molecular pathways modulated by fludarabine.
Indeed, in an effort to elucidate the mechanisms underlying the differential expression of these miRNAs, we analyzed the molecular pathways modulated by fludarabine in sensitive and refractory CLLs that emerged by mRNA expression analysis. The discovery that many p53 pathway genes (i.e. CDKN1A, BBC3/PUMA, PCNA, GADD45A) were activated in CRs but not in NRs provides further support to the notion that fludarabine treatment of patients with CLL induces a p53-dependent gene expression response  and that a defective p53 pathway in refractory CLLs, due to 17p deletions, p53 mutations and other p53-linked dysfunctions, is associated with fludarabine resistance [4, 25, 26, 28]. It is therefore possible that the differential expression of miRNAs between CR versus NR patients may be affected by a defective p53 pathway. Interestingly, it has been reported that miR-221/222 cluster is repressed by p53 , implicating that a non-functional p53 pathway may lead to its up-regulation, as detected in NR patients. An example of an aberrantly expressed miRNA influenced by p53 in fludarabine-refractory CLL has been recently reported by Zenz and colleagues, who observed a higher frequency of fludarabine refractory CLLs in the group of low-expressing miR-34a, than in similar patients with high expression of this miRNA . MiR-34a was shown to be a direct transcriptional target of p53 . Although we did not find a significant differential expression level of miR-34a in NR compared with CR CLLs, our data concur with those of Zenz et al., in fact 3 patients with the 17p deletion involved in this study displayed no miR-34a expression (data not shown).
By comparing the miRNA expression profile of sensitive and refractory CLLs, we have been able to identify expression signatures that could correctly classify more than 80% of the CR vs NR cases, either before or after treatment. Interestingly, after validation by quantitative PCR of the most significant miRNAs (miR-21, miR-222 and miR-148a) in an independent population, the prediction of response to therapy for pre-therapy samples was impressive: a predictive score based on miRNA expression levels reached an overall accuracy of 100%. This information may have important clinical implications because it might spare subsequent treatment in patients identified as resistant during disease progression.
MiRNAs aberrant expression may not only represent a phenomenon able to reveal an underlying molecular defect, such as p53 dysfunction, but it may also represent one of the possible mechanisms responsible for fludarabine resistance. In this study, we found that some miRNAs, namely miR-222, miR-148a and miR-21 were differentially expressed between NR and CR CLLs both before and after fludarabine treatment and were all up-regulated in the NR group. These findings, not previously described in CLL, appears to be truly significant.
Indeed, miR-222 can target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57 and the c-KIT receptor (source: Tarbase) thus promoting cell cycle progression [23, 32]. Among the recently validated targets there are the BH3-only pro-apoptotic protein BMF , the cell adhesion molecule ICAM-1 , matrix metalloproteinase 1 (MMP1) and manganese superoxide dismutase 2 (SOD2), which are both involved in tongue carcinoma metastasis promotion  and whose expression is frequently altered in cancer. An higher expression of miR-222 has been associated with poor survival in pancreatic cancer  and the enforced expression of miR-221/222 improved the growth of prostate carcinoma xenografts in mice . Moreover, anti-sense inhibition of miR-221 could increase susceptibility to gemcitabine .
MiR-21 up-regulation appears to be an important mechanism of malignant transformation, since it was found up-regulated in several types of neoplasia (see Spizzo et al. for a list ); it accounts, among the known validated targets, numerous tumors suppressor genes, such as TPM1, PDCD4, maspin (SERPINB3) and PTEN (source: Tarbase). New interesting targets of miR-21 were recently identified through the validation of proteomic results . It was also correlated with poor prognosis in tongue squamous cell carcinoma , colon , breast  and pancreatic cancer . Interestingly, the use of the anti-miR-21 AMOs increases susceptibility of colangiocarcinoma cells to gemcitabine  and elicit a pro-apoptotic response in glioblastoma and breast cancer cells [46, 47].
Here, we report that inhibition of miR-222 and miR-21 in the MEG-01 human cells using an anti-miRNA oligonucleotide increases susceptibility to fludarabine, as shown by the significant increase in caspase activity. Our findings suggest a direct role of miR-222 and miR-21 in conferring resistance to fludarabine chemotherapy independently from the p53 pathway, which is dysfunctional in this human cell line .
Less clear is the role of miR-148a. MiR-148a is up-regulated in NR CLL patients either before and after fludarabine treatment and it was identified also as resistance predictor in pre CLLs. However, its mechanism of action is not clear, as only two gene targets are presently experimentally validated. One is the isoform 1 of DNA methyltransferase 3b (DNMT3b1)  and the other is the pregnane X receptor gene  whose repression, in turn, reduces the expression of CYP3A4 enzyme. Our assay revealed that its mechanism of action may not be linked to apoptosis inhibition in resistant patients. Therefore, its role in fludarabine resistance remains unknown.