**4. Modulation of miRNAs expression levels in ALL: a therapeutic approach**

Children diagnosed with ALL, in most cases follow a standard treatment protocol divided into three phases: induction, consolidation and maintenance, and sometimes a central nervous system-directed therapy. The drugs used in induction and consolidation phases usually include prednisolone (PRED), vincristine (VCR), L-asparaginase (L-ASP) and daunorubicin (DNR), all these drugs presents late effects. The maintenance phase is the most prolongated treatment in childhood ALL, however, it involves a much less intensive regimen than induction and consolidation phases. The maintenance phase has been demonstrated to lower the

risk of relapse, but could be a cause for emergence new mutations leading to drug resistance [16]. Even though the great effort in the development of new treatment strategies, drug resistance is the major cause of chemotherapy failure and relapse in pediatric patients. The classification of patients resistant or sensitive to drugs can be carried out based on the expression level of miRNAs.

The multidrug resistance (MDR) represents a problem in the treatment of pediatric ALL. It is considered an ubiquitous and severe clinical problem. MDR is mediated by adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Several chemotherapeutic drugs are actively transported by ABC transporters across the cellular membranes leading to chemotherapy failure. Recent evidence indicates that some miRNAs tend to enhance chemotherapy response by modulating the expression of ABC transporters. In a study, the transfection of miR-326 into HepG2 cells demonstrated enhanced response to chemotherapeutic drugs due to downregulation of ABC transporter ABCC144 [16, 39]. Previous reports indicated that overexpression of ABCA2 and ABCA3 genes increases the risk of MDR and relapse in pediatric ALL patients [40]. Based upon bioinformatics analysis, miR-326 was identified as negative regulator of MDR-related genes, ABCA2 and ABCA3 in particular. Further evaluation of the miR-326 expression levels in pediatric ALL patients resistant to chemotherapy (MDR+) revealed that miR-326 is significantly decreased in MDR+ compared to the MDR- control group, supporting the idea that low level expression of miR-326 impacts directly on chemotherapy treatment response [41, 42].

Glucocorticoids (GC) are a group of drugs clinically used to treat ALL due to their involvement in cell progression, immunoglobulin, lymphokine production and apoptosis in immature lymphoblasts. The MLL rearrangements are common genetic abnormality in ALL. MLL-AF4 is the result of a balanced translocation between MLL and AF4 and it occurs in approximately 50% of ALL cases in infants. MLL-AF4 is an indicative of poor prognosis due to failure in GC-induced apoptosis. miR-128b and miR-221 are downregulated in this type of ALL and leads to overexpression of their targets: CDKN1B, MLL, AF4 and both MLL-AF4 and AF4-MLL fusion genes (involved in AL development). The restoration of miR-128b and miR-221 results in the downregulation of their aforementioned targets and increase the sensitivity to GC therapy [37, 43]. After transiently overexpression of miR-17 in a pre-B cell line (SUP-B15), it was found a reduced dexamethasone (DEX)-induced apoptosis indicating resistance to DEX. Further inhibition of miR-17 by locked nucleic acid inhibitor enhanced the response to DEX. The development of miRNA inhibitors, antagomiRs and agomiRs allows to modulate miRNAs levels in order to obtain a better response in ALL treatment.

AntagomiR is a chemically-modified single-strand miRNA inhibitor used to block miRNA regulation of target gene expression efficiently. AntagomiRs are synthesized to reduce the ability of endogenous miRNAs to silence target mRNA transcripts. They can downregulate the corresponding endogenous miRNAs by either local or systemic injection into the animals. Their structure consists of a single-strand RNA carrying the chemically modifications functioning by blocking a target of miRNA. The strand of the antagomiR has 2 phosphorothionates at the 5'end, 4 phosphorothionates, 1 cholesterol group at the 3'end and full-length nucleotide 2′-methoxy modification. Its stability is significantly higher than miRNA inhibitors. On the other hand, agomiR is a chemically-modified double-strand miRNA mimics which can mimic mature endogenous miRNAs after transfection into cells. They can upregulate the endogenous miRNA activity by utilizing the natural miRNA machinery. The antisense strand of the agomiR has 2 phosphorothionates at the 5'end, 4 phosphorothionates, 1 cholesterol group at the 3'end and full-length nucleotide 2′-methoxy modification. The chemically-modified structure

**115**

*MicroRNAs and Their Role in Acute Lymphoblastic Leukemia*

recommended for miRNA functional *in vitro* and *in vivo* studies.

sion of miR-101 may be related to chemoresistance in T-ALL [44].

of agomiR increases its stability and activity. Both, agomiRs and antagomiRs are

Once a miRNA has been established or identified as a target to be downregulated, or up regulated, agomiRs or antagomiRs can be used to modulate miRNAs levels and further determination of anticancer effects. This is a promising and interesting approach to combine miRNA-based therapy and current chemotherapy to synergistically improve clinical outcomes. The potential therapeutic miRNAs targets described below can be modulated by using agomiR or antagomiR. The miR-101 has a potential role in drug response to doxorubicin (DOXO) in Jurkat T-ALL. It was shown that miR-101 targets NOTCH1 which is linked to enhanced DOXO sensitivity to myeloma cell lines in mouse. Therefore, miR-101 may enhance DOXO-mediated apoptosis by repressing NOTCH1, while low expres-

For ALL patients with BCR-ABL fusion gene, the treatment with tyrosine-kinase inhibitors (TKI) could be a promising strategy even though the prognosis is suboptimal. BCR-ABL1 and ABL1 are direct targets of miR-203. However, miR-203 is silenced by genetic and epigenetic mechanisms in hematopoietic malignancies as leukemia. It has been reported that the restoration of miR-203 expression reduces BCR-ABL1 and ABL1 levels in cells and leads to arrest in cell proliferation [15, 45]. In addition, the inhibition of the DNMT3A gene, which gives the instructions for making the enzyme DNA methyltransferase alpha involved in DNA methylation and gene silencing, can be assessed by increasing miR-217 or mimics expression. This may prevent drug resistance to TKI in Philadelphia-chromosome-positive ALL patients, being another therapeutic strategy [46]. According to the aforementioned, the demethylation may be a potential therapeutic strategy in ALL. As mentioned before, the miR-143 is epigenetically repressed by promoter hypermethylation in MLL-AF4-positive primary blast, but not in normal bone marrow cells, and neither in MLL-AF4-negative primary blasts. Besides, MLL-AF4 expression is regulated by miR-143, such not being possible in MLL-AF4-positive cells. The restoration of miR-143 levels could induce apoptosis and regulate in a negative way the of the

The miR-125b-2 cluster, consisting of miR-125b, miR-99a and let-7c, is increased in ETV6-RUNX1+ leukemia. According to previous reports, the miR-125b-2 cluster expression is not regulated by the ETV6-RUNX1 fusion protein, which indicates that the expression of this cluster may be an independent leukemia

led to increased cell sensitivity to DOXO and staurosporine treatment. Hence, the overexpression of miR-125b-2 cluster confers to leukemic cells survival advantage through the inhibition of apoptosis and failure in activation of caspase-3 [48]. These findings support the idea that miR-125b-2 cluster is a potential therapeutic

In pediatric ALL patients, a genome-wide miRNA analysis indicated a reduced miR-335 expression, such being the most significant miRNA abnormality associated with a poor outcome. Furthermore, the overexpression of miR-335 significantly sensitized the ALL cells to PRED. The role of miR-335 in PRED resistance was studied by investigating downstream pathways. The results suggested that low level of miR-335 leads to higher MAPK1-mediated survival. Further treatment with MEK/ERK inhibitor enhanced PRED-induced cell death suggesting that using synthetic miR-335 and overriding MAPK1 activity plus MEK/ERK pathway inhibition may provide a promising therapeutic strategy to overcome PRED resistance [49]. In regards of T-ALL, few studies investigate the role of miRNAs in GC response.

GC bind to its receptor (GCR), which acts as a ligand-dependent-transcription factor, inducing cell cycle arrest and apoptosis in cancer cells [50]. The GCR is

cell line Reh

event. Further knockdown of miR-125b-2 cluster in the *ETV6-RUNX1*<sup>+</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.94960*

leukemia cells growth [47].

target in pediatric ALL.

#### *MicroRNAs and Their Role in Acute Lymphoblastic Leukemia DOI: http://dx.doi.org/10.5772/intechopen.94960*

*Acute Leukemias*

response [41, 42].

obtain a better response in ALL treatment.

risk of relapse, but could be a cause for emergence new mutations leading to drug resistance [16]. Even though the great effort in the development of new treatment strategies, drug resistance is the major cause of chemotherapy failure and relapse in pediatric patients. The classification of patients resistant or sensitive to drugs can be

The multidrug resistance (MDR) represents a problem in the treatment of pediatric ALL. It is considered an ubiquitous and severe clinical problem. MDR is mediated by adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Several chemotherapeutic drugs are actively transported by ABC transporters across the cellular membranes leading to chemotherapy failure. Recent evidence indicates that some miRNAs tend to enhance chemotherapy response by modulating the expression of ABC transporters. In a study, the transfection of miR-326 into HepG2 cells demonstrated enhanced response to chemotherapeutic drugs due to downregulation of ABC transporter ABCC144 [16, 39]. Previous reports indicated that overexpression of ABCA2 and ABCA3 genes increases the risk of MDR and relapse in pediatric ALL patients [40]. Based upon bioinformatics analysis, miR-326 was identified as negative regulator of MDR-related genes, ABCA2 and ABCA3 in particular. Further evaluation of the miR-326 expression levels in pediatric ALL patients resistant to chemotherapy (MDR+) revealed that miR-326 is significantly decreased in MDR+ compared to the MDR- control group, supporting the idea that low level expression of miR-326 impacts directly on chemotherapy treatment

Glucocorticoids (GC) are a group of drugs clinically used to treat ALL due to their involvement in cell progression, immunoglobulin, lymphokine production and apoptosis in immature lymphoblasts. The MLL rearrangements are common genetic abnormality in ALL. MLL-AF4 is the result of a balanced translocation between MLL and AF4 and it occurs in approximately 50% of ALL cases in infants. MLL-AF4 is an indicative of poor prognosis due to failure in GC-induced apoptosis. miR-128b and miR-221 are downregulated in this type of ALL and leads to overexpression of their targets: CDKN1B, MLL, AF4 and both MLL-AF4 and AF4-MLL fusion genes (involved in AL development). The restoration of miR-128b and miR-221 results in the downregulation of their aforementioned targets and increase the sensitivity to GC therapy [37, 43]. After transiently overexpression of miR-17 in a pre-B cell line (SUP-B15), it was found a reduced dexamethasone (DEX)-induced apoptosis indicating resistance to DEX. Further inhibition of miR-17 by locked nucleic acid inhibitor enhanced the response to DEX. The development of miRNA inhibitors, antagomiRs and agomiRs allows to modulate miRNAs levels in order to

AntagomiR is a chemically-modified single-strand miRNA inhibitor used to block miRNA regulation of target gene expression efficiently. AntagomiRs are synthesized to reduce the ability of endogenous miRNAs to silence target mRNA transcripts. They can downregulate the corresponding endogenous miRNAs by either local or systemic injection into the animals. Their structure consists of a single-strand RNA carrying the chemically modifications functioning by blocking a target of miRNA. The strand of the antagomiR has 2 phosphorothionates at the 5'end, 4 phosphorothionates, 1 cholesterol group at the 3'end and full-length nucleotide 2′-methoxy modification. Its stability is significantly higher than miRNA inhibitors. On the other hand, agomiR is a chemically-modified double-strand miRNA mimics which can mimic mature endogenous miRNAs after transfection into cells. They can upregulate the endogenous miRNA activity by utilizing the natural miRNA machinery. The antisense strand of the agomiR has 2 phosphorothionates at the 5'end, 4 phosphorothionates, 1 cholesterol group at the 3'end and full-length nucleotide 2′-methoxy modification. The chemically-modified structure

carried out based on the expression level of miRNAs.

**114**

of agomiR increases its stability and activity. Both, agomiRs and antagomiRs are recommended for miRNA functional *in vitro* and *in vivo* studies.

Once a miRNA has been established or identified as a target to be downregulated, or up regulated, agomiRs or antagomiRs can be used to modulate miRNAs levels and further determination of anticancer effects. This is a promising and interesting approach to combine miRNA-based therapy and current chemotherapy to synergistically improve clinical outcomes. The potential therapeutic miRNAs targets described below can be modulated by using agomiR or antagomiR.

The miR-101 has a potential role in drug response to doxorubicin (DOXO) in Jurkat T-ALL. It was shown that miR-101 targets NOTCH1 which is linked to enhanced DOXO sensitivity to myeloma cell lines in mouse. Therefore, miR-101 may enhance DOXO-mediated apoptosis by repressing NOTCH1, while low expression of miR-101 may be related to chemoresistance in T-ALL [44].

For ALL patients with BCR-ABL fusion gene, the treatment with tyrosine-kinase inhibitors (TKI) could be a promising strategy even though the prognosis is suboptimal. BCR-ABL1 and ABL1 are direct targets of miR-203. However, miR-203 is silenced by genetic and epigenetic mechanisms in hematopoietic malignancies as leukemia. It has been reported that the restoration of miR-203 expression reduces BCR-ABL1 and ABL1 levels in cells and leads to arrest in cell proliferation [15, 45]. In addition, the inhibition of the DNMT3A gene, which gives the instructions for making the enzyme DNA methyltransferase alpha involved in DNA methylation and gene silencing, can be assessed by increasing miR-217 or mimics expression. This may prevent drug resistance to TKI in Philadelphia-chromosome-positive ALL patients, being another therapeutic strategy [46]. According to the aforementioned, the demethylation may be a potential therapeutic strategy in ALL. As mentioned before, the miR-143 is epigenetically repressed by promoter hypermethylation in MLL-AF4-positive primary blast, but not in normal bone marrow cells, and neither in MLL-AF4-negative primary blasts. Besides, MLL-AF4 expression is regulated by miR-143, such not being possible in MLL-AF4-positive cells. The restoration of miR-143 levels could induce apoptosis and regulate in a negative way the of the leukemia cells growth [47].

The miR-125b-2 cluster, consisting of miR-125b, miR-99a and let-7c, is increased in ETV6-RUNX1+ leukemia. According to previous reports, the miR-125b-2 cluster expression is not regulated by the ETV6-RUNX1 fusion protein, which indicates that the expression of this cluster may be an independent leukemia event. Further knockdown of miR-125b-2 cluster in the *ETV6-RUNX1*<sup>+</sup> cell line Reh led to increased cell sensitivity to DOXO and staurosporine treatment. Hence, the overexpression of miR-125b-2 cluster confers to leukemic cells survival advantage through the inhibition of apoptosis and failure in activation of caspase-3 [48]. These findings support the idea that miR-125b-2 cluster is a potential therapeutic target in pediatric ALL.

In pediatric ALL patients, a genome-wide miRNA analysis indicated a reduced miR-335 expression, such being the most significant miRNA abnormality associated with a poor outcome. Furthermore, the overexpression of miR-335 significantly sensitized the ALL cells to PRED. The role of miR-335 in PRED resistance was studied by investigating downstream pathways. The results suggested that low level of miR-335 leads to higher MAPK1-mediated survival. Further treatment with MEK/ERK inhibitor enhanced PRED-induced cell death suggesting that using synthetic miR-335 and overriding MAPK1 activity plus MEK/ERK pathway inhibition may provide a promising therapeutic strategy to overcome PRED resistance [49].

In regards of T-ALL, few studies investigate the role of miRNAs in GC response. GC bind to its receptor (GCR), which acts as a ligand-dependent-transcription factor, inducing cell cycle arrest and apoptosis in cancer cells [50]. The GCR is

modulated by miR-142-3p, which represses GCR biosynthesis. The inhibition of miR-142-3p in T-ALL cell lines resulted in increased GC-mediated cell death compared to not inhibited cells. Another protein involved in GC resistance via GCR repression is the FKBP51, which is negatively regulated by miR-100 and miR-99. Both miRNAs are found to be downregulated in the T-ALL as well as the B-ALL. In transfected T-ALL cell lines with mimics of miR-100 and miR-99, the result led to enhanced sensitivity to CG and apoptosis. In addition, previous studies reported that miR-100 and miR-99 act synergistically with miR-125 enhancing resistance to VCR [51, 52]. *In vitro* studies indicate that only the overexpression of the three aforementioned miRNAs led to VCR resistance. Moreover, hypoxia is a crucial microenvironmental factor supporting self-renewal of leukemic stem cells in bone marrow niches. miR-210 is one of the hypoxia-regulated miRNAs most studied in cancer including its role in drug resistance and as a prognostic potential. The miR-210 levels were modified in ETV6-RUNX+ Reh and MLL-AF4 + RS4; 11 pediatric ALL cell lines by introducing antagomiR and agomiR to miR-210. After 24 h of the transfection, both cell lines were treated with the DNR, VCR, DEX and L-ASP, individually or in combinations of the four drugs. In ETV6-RUNX+ Reh cells, the half maximal inhibitory concentrations (IC50s) of DNR, DEX and L-ASP were significantly decreased (in agomiR-transfected cells) or increased (in antagomiR-transfected cells) compared to cells transfected with negative control mimics. At the other extreme, the IC50s of DNR, DEX and VCR were decreased (in agomiR-transfected cells) or increased (in antagomiR-transfected cells) in MLL-AF4 + RS4;11 cells [53]. In regards with these findings, the use of agomiRs and antagomiRs to miRNAs could be an alternative to overcome resistance to chemotherapy. Nevertheless, the great challenge in clinical and nonclinical trials, is the delivery of synthetic miRNAs to leukemia cells effectively.
