**Abstract**

Acute lymphoblastic leukemia (ALL) has been established as the most common acute leukemia in children, accounting for 80–85% of cases. ALL occurs mostly in children and it is considered as a high-risk disease in the elderlies. ALL is characterized by a clonal disorder where the normal hematopoiesis is replaced by a malignant clonal expansion of lymphoid progenitors. Although many therapeutic strategies have been established to treat ALL leading to improved survival rates, the short-term and long-term complications derived from treatment toxicity represent a critical risk for patients. The treatment-related toxicity suggests a need for the development of new therapy strategies to effectively treat high-risk and low-risk disease. Nowadays, an important approach is focused on the identification of molecules involved in the mechanisms that lead to leukemia generation and progression to determine potential targets at the transcriptional level. MicroRNAs (miRNAs) are a group of key molecules that regulate signaling pathways related to lymphopoiesis. miRNAs participate in the regulation of hematopoietic differentiation and proliferation, as well as their activity. The present review details the recompilation of evidences about the relation between miRNAs and lymphopoiesis, ALL development and progression in order to propose and explore novel strategies to modulate ALL-related miRNA levels as a therapeutic approach.

**Keywords:** miRNAs, therapeutics, lymphopoiesis, acute lymphoblastic leukemia, agomiR, antagomiR

### **1. Acute lymphoblastic leukemia generalities**

Acute lymphoblastic leukemia (ALL) has been established as the most common acute leukemia in children representing 80–85% of cases [1]. The global incidence ranges from three to five cases per 100, 000 population and the lowest incidence occurs in the adults older than 65 years old [2]. Even though ALL occurs mostly in children, it represents a devastating disease in the elderlies. ALL is a clonal disorder where the normal hematopoiesis is replaced by a malignant clonal expansion of lymphoid progenitors (blasts) [3]. Such clonal disorder is characterized by genetic abnormalities that lead to a block in B or T cell differentiation leading to abnormal cell proliferation and apoptosis in the bone marrow, peripheral blood and extramedullary sites [4, 5]. The overall survival of patients with ALL has been improved after treatment stratification according to immunophenotype and genotype. This treatment has allowed to incorporate, into therapy protocols, and more effective drug combinations [6]. Although improved survival rates have been obtained, the short-term and long-term complications derived from treatment toxicity represent

a critical risk for patients. This suggests a need for the development of new therapy strategies to diminish the treatment-related toxicity and effectively treat high-risk and low-risk disease. An important approach is focused on the mechanisms that lead to leukemia generation and progression by identifying potential targets at the transcriptional level.

The development of B-lymphocytes and T-lymphocytes from a lymphoid progenitor occurs in the bone marrow and thymus, respectively, and the normal lymphopoiesis is regulated at a transcriptional level involving transcription factors acting as regulators of genes expression [7]. Nevertheless, a question arises: does the transcriptional process is sufficient to control the cell fate, lineage and other aspects of cellular functioning? The answer is "no", and there are other key mechanisms involved. MicroRNAs (miRNAs) are a group of key molecules that regulate signaling pathways related to lymphopoiesis. miRNAs regulate hematopoietic differentiation and proliferation, as well as their activity. Besides, a deregulation in miRNAs expression is observed in leukemias having a main role in the pathogenesis of such clonal disorder [7, 8]. The first connection between miRNAs and leukemia indicated that 13q14 deletion in B-cell chronic lymphocytic leukemias induced the loss of miR-16-1 and miR-15a in 70% of chronic lymphocytic leukemias [9]. In addition, recent studies suggested that at least 50% of miRNAs are located in cancerassociated genomic regions and mRNAs are regulated by these miRNAs populations [5]. Moreover, different patterns of miRNAs are observed in normal lymphopoiesis and leukemias. The investigation of the relation between miRNAs and pathological cellular processes represents an interesting approach for the development of new therapeutic strategies.

## **2. miRNAs biology**

miRNAs are endogenous small non-coding RNAs with a length of 19–25 nucleotides (nt) and their genes are transcribed by RNA polymerase II (Pol II) in the nucleus [10, 11]. miRNAs transcripts are first generated as pri-miRNAs structures (up to 1000 nt of length) which contain cap structures and poly-A tails (**Figure 1**). The pri-miRNAs transcript is briefly processed into precursor miRNA (pre-miRNA) transcript of 60–120 nt of length by the microprocessor complex formed by class 2 ribonuclease III enzyme called DROSHA and the DiGeorge Syndrome Critical Region Gene (DGCR8) protein. The pre-miRNA structure bears a hairpin structure with 2-nt overhang at the 3'end [12, 13]. The next step consists in exporting the pre-miRNA from the nucleus to the cytosol. This process is mediated by the exportin-5 (a RanGTP-dependent dsRNA-binding protein) which recognizes the overhang in the pre-miRNA as a target to be transported [14].

**111**

receptor [24, 25].

*MicroRNAs and Their Role in Acute Lymphoblastic Leukemia*

Once in the cytosol, and pre-miRNA is processed by the DICER enzyme (RNAse type III) leading to a mature miRNA duplex (miRNA: miRNA) about 22 nt of length. The miRNA duplex is loaded into the RNA-induced silencing complex (RISC), composed by of the Argonaute2 (Ago2) and the trans-activator RNA (tar)-binding protein (TRBP). Note that the mature single-strand miRNA is retained by the Argo2 protein in the RISC complex to guide the gene silencing by binding to its mRNA target leading to mRNA degradation [10, 13, 15]. Recent evidence indicates that some pri-miRNAs contain open reading fragments about 300 nt and could be transported, without being processed, into the cytosol, and then become translated into micropeptides, having the ability to influence a variety of cellular processes. This is an additional field of opportunity to carry out functional studies with the aim to develop novel therapeutic strategies [16].

**3. miRNAs and their implications in lymphopoiesis and ALL** 

different according to normal or malignant lymphopoietic process.

Lymphopoiesis is a process in which hematopoietic stem cells (HSCs) differentiate into an immature form called lymphoid progenitor and finally into mature B- or T-lymphocytes [17]. The miRNAs play a key role in the process of cell differentiation by regulating several signaling pathways. The pattern of miRNAs expression is

The miRNA-150 (miR-150) is expressed in B- and T-cells and the lymphoid progenitors express the miR-150 to promote B-cell maturation and by assisting in the transition of the progenitor B-cells (pro-B) to precursor B-cell (pre-B) stage. When a premature miR-150 expression occurs, the result is a blocked transition from pro-B to pre-B stage [18, 19]. In the thymus, the expression of miR-150 enhances T-cell development and its key pathways (Notch pathway) and prevents an alternative lineage differentiation (B-cell differentiation) in the progenitors. Since C-myb is an essential transcription factor involved in the early lymphoid development, a downregulation eventually leads to an arrest from pro-B to pre-B stage. This event is

The miRNA-155 (miR-155) is upregulated in both B- and T-cells in their activated and mature stage. In the case of B-cells differentiation, miR-155 inhibits PU.1 expression leading to Pax5 downregulation and the initiation of the plasma cell differentiation pathway. This event is known as the miR-155-PU.1 axis. The role of miR-155 in T-cells differentiation depends on the level of miR-155 expression. The expression of miR-155 mediates the differentiation of T-cells into T-helper type 1,

The miRNA-181 (miR-181) is a family of miRNAs and is composed of three clusters located in a different chromosome. Hsa-miR-181a-1 and hsa-miR181b-1 are located on chromosome 1; hsa-miR-181a-2 and hsa-miR181-b-2 are located on chromosome 9 and finally hsa-miR181c-1 and hsa-miR-181c-2 are located on chromosome19 [22]. miR-181 family have been reported to be implicated in lung and breast cancers [23]. miR-181a can act as inhibitor of the normal cellular response to DNA damage by affecting the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). In the early stage of B-cells differentiation, the expression of miR-181 is high and decreases subsequently within differentiation [17], and during T-cell development, the miR-181 is highly expressed in double-positive T-cells. The targets for miR-181 are BCL-2, CD89, EGR1 and T-cell

while its absence leads to T-cells transition into T-helper type [20, 21].

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

**3.1 Lymphopoiesis regulation by miRNAs**

accompanied by miR-150 overexpression [15, 17].

**development**

**Figure 1.** *miRNAs biogenesis and gene silencing mediated by miRNA.*

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

*Acute Leukemias*

transcriptional level.

therapeutic strategies.

**2. miRNAs biology**

a critical risk for patients. This suggests a need for the development of new therapy strategies to diminish the treatment-related toxicity and effectively treat high-risk and low-risk disease. An important approach is focused on the mechanisms that lead to leukemia generation and progression by identifying potential targets at the

The development of B-lymphocytes and T-lymphocytes from a lymphoid progenitor occurs in the bone marrow and thymus, respectively, and the normal lymphopoiesis is regulated at a transcriptional level involving transcription factors acting as regulators of genes expression [7]. Nevertheless, a question arises: does the transcriptional process is sufficient to control the cell fate, lineage and other aspects of cellular functioning? The answer is "no", and there are other key mechanisms involved. MicroRNAs (miRNAs) are a group of key molecules that regulate signaling pathways related to lymphopoiesis. miRNAs regulate hematopoietic differentiation and proliferation, as well as their activity. Besides, a deregulation in miRNAs expression is observed in leukemias having a main role in the pathogenesis of such clonal disorder [7, 8]. The first connection between miRNAs and leukemia indicated that 13q14 deletion in B-cell chronic lymphocytic leukemias induced the loss of miR-16-1 and miR-15a in 70% of chronic lymphocytic leukemias [9]. In addition, recent studies suggested that at least 50% of miRNAs are located in cancerassociated genomic regions and mRNAs are regulated by these miRNAs populations [5]. Moreover, different patterns of miRNAs are observed in normal lymphopoiesis and leukemias. The investigation of the relation between miRNAs and pathological cellular processes represents an interesting approach for the development of new

miRNAs are endogenous small non-coding RNAs with a length of 19–25 nucleotides (nt) and their genes are transcribed by RNA polymerase II (Pol II) in the nucleus [10, 11]. miRNAs transcripts are first generated as pri-miRNAs structures (up to 1000 nt of length) which contain cap structures and poly-A tails (**Figure 1**). The pri-miRNAs transcript is briefly processed into precursor miRNA (pre-miRNA) transcript of 60–120 nt of length by the microprocessor complex formed by class 2 ribonuclease III enzyme called DROSHA and the DiGeorge Syndrome Critical Region Gene (DGCR8) protein. The pre-miRNA structure bears a hairpin structure with 2-nt overhang at the 3'end [12, 13]. The next step consists in exporting the pre-miRNA from the nucleus to the cytosol. This process is mediated by the exportin-5 (a RanGTP-dependent dsRNA-binding protein) which recognizes the overhang in the pre-miRNA as a target to be transported [14].

**110**

**Figure 1.**

*miRNAs biogenesis and gene silencing mediated by miRNA.*

Once in the cytosol, and pre-miRNA is processed by the DICER enzyme (RNAse type III) leading to a mature miRNA duplex (miRNA: miRNA) about 22 nt of length. The miRNA duplex is loaded into the RNA-induced silencing complex (RISC), composed by of the Argonaute2 (Ago2) and the trans-activator RNA (tar)-binding protein (TRBP). Note that the mature single-strand miRNA is retained by the Argo2 protein in the RISC complex to guide the gene silencing by binding to its mRNA target leading to mRNA degradation [10, 13, 15]. Recent evidence indicates that some pri-miRNAs contain open reading fragments about 300 nt and could be transported, without being processed, into the cytosol, and then become translated into micropeptides, having the ability to influence a variety of cellular processes. This is an additional field of opportunity to carry out functional studies with the aim to develop novel therapeutic strategies [16].

### **3. miRNAs and their implications in lymphopoiesis and ALL development**

#### **3.1 Lymphopoiesis regulation by miRNAs**

Lymphopoiesis is a process in which hematopoietic stem cells (HSCs) differentiate into an immature form called lymphoid progenitor and finally into mature B- or T-lymphocytes [17]. The miRNAs play a key role in the process of cell differentiation by regulating several signaling pathways. The pattern of miRNAs expression is different according to normal or malignant lymphopoietic process.

The miRNA-150 (miR-150) is expressed in B- and T-cells and the lymphoid progenitors express the miR-150 to promote B-cell maturation and by assisting in the transition of the progenitor B-cells (pro-B) to precursor B-cell (pre-B) stage. When a premature miR-150 expression occurs, the result is a blocked transition from pro-B to pre-B stage [18, 19]. In the thymus, the expression of miR-150 enhances T-cell development and its key pathways (Notch pathway) and prevents an alternative lineage differentiation (B-cell differentiation) in the progenitors. Since C-myb is an essential transcription factor involved in the early lymphoid development, a downregulation eventually leads to an arrest from pro-B to pre-B stage. This event is accompanied by miR-150 overexpression [15, 17].

The miRNA-155 (miR-155) is upregulated in both B- and T-cells in their activated and mature stage. In the case of B-cells differentiation, miR-155 inhibits PU.1 expression leading to Pax5 downregulation and the initiation of the plasma cell differentiation pathway. This event is known as the miR-155-PU.1 axis. The role of miR-155 in T-cells differentiation depends on the level of miR-155 expression. The expression of miR-155 mediates the differentiation of T-cells into T-helper type 1, while its absence leads to T-cells transition into T-helper type [20, 21].

The miRNA-181 (miR-181) is a family of miRNAs and is composed of three clusters located in a different chromosome. Hsa-miR-181a-1 and hsa-miR181b-1 are located on chromosome 1; hsa-miR-181a-2 and hsa-miR181-b-2 are located on chromosome 9 and finally hsa-miR181c-1 and hsa-miR-181c-2 are located on chromosome19 [22]. miR-181 family have been reported to be implicated in lung and breast cancers [23]. miR-181a can act as inhibitor of the normal cellular response to DNA damage by affecting the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). In the early stage of B-cells differentiation, the expression of miR-181 is high and decreases subsequently within differentiation [17], and during T-cell development, the miR-181 is highly expressed in double-positive T-cells. The targets for miR-181 are BCL-2, CD89, EGR1 and T-cell receptor [24, 25].


**113**

**approach**

*MicroRNAs and Their Role in Acute Lymphoblastic Leukemia*

The miR-17-92 cluster is composed of six miRNAs including miR-17, miR-18a, miR-19a, miR19-b-1, miR-20a and miR-92-1 and is highly expressed in both B- and T-lymphoid progenitors and subsequently decreases within maturation [27]. When miR-17-92 cluster is downregulated or absent, occurs B-cell differentiation disorder due to the overexpression of the proapoptotic protein BIM (target of miR-17-92 cluster). On the other hand, when miR-12-92 cluster is overexpressed during lymphopoiesis, promotes lymphoproliferative disorders and severe autoimmunity [36]. The aforementioned miRNAs play a critical role on the lymphopoiesis and the change of their expression pattern leads to blood disorders or malignant lymphopoiesis mechanisms. Such is the reason why miRNAs can be potential targets for

Besides the involvement of aberrant miRNAs expression in malignant lymphopoiesis, it may be related to drug resistance mechanisms. Focusing on promoting the reversal expression of such miRNA may enhance disease management and improve clinical outcomes. By comparing miRNAs expression in both normal pediatric bone marrow and pediatric ALL, an overexpression of miR-100, miR-196b in ALL and lower expression of let-7e in normal bone marrow were reported [37]. In a study conducted in childhood B-ALL and T-ALL, the miRNA expression profile in the bone marrow was evaluated to discriminate between T-ALL and B-ALL. The results indicated a downregulation of miR-708-5p, miR-497-5p, miR-151a-5p, miR-151b, miR-371b-5p, miR-455-5p, miR-195-5p, miR-1266-5p, miR-574-5p, miR-425-5p and upregulation of miR-450b-5p, miR-450a-5p, miR-542-5p, miR-424-5p, miR-629-5p, miR-29c-5p in T-ALL. Further machine process analyses showed that miR-29c-5p, involved in calcium signaling, is involved in B-cell fate and it is the best discriminator to stablish childhood B-ALL or T-ALL. In addition, clinical samples of T-ALL were studied and reveled that miR-223, miR-19b, miR-20a, miR-92, miR-142-3p, miR-150, miR-93, miR-26a, miR-16, and miR-342., miR-19b,-20a,-26a,-92 and miR-223 target T-ALL tumor suppressors, such as IKAROS, PTEN, BIM, PHF6, NF1 and FBXW7 [37], which means that overexpression of such miRNAs could lead

to ALL development and represents a potential therapeutic target.

The identification of miRNAs that target genes of MYC, TFs of CDX2 and IncRNA of XIST could represent an approach to ALL therapeutics, due to that the aforementioned genes may play important role in the development of B-ALL. NOTCH1 is another important target due to mutations of gain of function, which are implicated in T-ALL development as well as NOTCH1 and NOTCH2 that take part in B-malignances [38]. **Table 1** exhibits a list of miRNAs involved in ALL development and pathogenesis considered as potential therapeutic targets.

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

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

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

treatment, diagnosis and prognosis in ALL.

**3.2 The role of miRNAs in ALL development**

#### **Table 1.**

*Function, targets and expression level of miRNAs related to ALL.*

*Acute Leukemias*

miR-150 Transition from pro-B to pre-B stage

1 cells

miR-181 B-cell differentiation and T-cell development

miR-17-92 Transition from pro-B to pre-B stage

miR-223 Reduction in cell growth and T-ALL development

miR-21 Regulation of PI3K/Akt/ mTOR pathway

miR-99a Promotes the differentiation

miR-100 Promotes the differentiation

monocytes cells

monocytes cells

of human granulocytes and

of human granulocytes and

miR-30a B-ALL development NOTCH1,

*Function, targets and expression level of miRNAs related to ALL.*

miR-19 T-ALL development PTEN, HOXA,

miR-19b T-ALL development IKAROS, PTEN,

miR-20a T-ALL development IKAROS, PTEN,

miR-92 T-ALL development IKAROS, PTEN,

miR-26a T-ALL development KAROS, PTEN,

differentiation into Th type

miR-155 Promotes T-cell

**miRNA Function Target miRNA** 

**expression in ALL vs. normal samples**

C-MYB High He *et al*.,

BCL-2, MYC, CDX2, CD69, GR1, and T-cell receptor

YLD, HOXA9, BIM, RUNX1, MYC

IKAROS, PTEN, BIM, PHF6, NF1, and FBXW7

BIM, PHF6, NF1, and FBXW7

BIM, PHF6, NF1, and FBXW7

BIM, PHF6, NF1, and FBXW7

BIM, PHF6, NF1, and FBXW7

mTOR, IGF-1, and MCL1

mTOR, IGF-1, and MCL1

CYLD, BIM, and NOTCH1

NOTCH2 and MYC

miR-29a T-ALL development MYC and CDX2 Low Zaidi et al.,

miR-123a PI3K signaling activation PTEN High Bertacchini

miR-126 B-ALL development MYC and CDX2 High Fulci et al.,

PTEN High Seddiki et al.,

**References**

2015 [26]

2014 [21]

2015 [24]

2015 [27]

2009 [28]

2011 [29]

2011 [29]

2011 [29]

2011 [29]

2011 [29]

et al., 2015 [30]

et al., 2015 [30]

[31], Li et al., 2013 [32]

et al., 2011 [33]

2013 [32]

2017 [34]

2015 [35]

High Veruci et al.,

High Rao et al.,

Low Mavrakis et al.,

High Mavrakis et al.,

High Mavrakis et al.,

High Mavrakis et al.,

High Mavrakis et al.,

Low Li et al., 2012

Low Li et al.

High Tsuchida

High Ortega et al.,

PTEN High Bertacchini

**112**

**Table 1.**

The miR-17-92 cluster is composed of six miRNAs including miR-17, miR-18a, miR-19a, miR19-b-1, miR-20a and miR-92-1 and is highly expressed in both B- and T-lymphoid progenitors and subsequently decreases within maturation [27]. When miR-17-92 cluster is downregulated or absent, occurs B-cell differentiation disorder due to the overexpression of the proapoptotic protein BIM (target of miR-17-92 cluster). On the other hand, when miR-12-92 cluster is overexpressed during lymphopoiesis, promotes lymphoproliferative disorders and severe autoimmunity [36].

The aforementioned miRNAs play a critical role on the lymphopoiesis and the change of their expression pattern leads to blood disorders or malignant lymphopoiesis mechanisms. Such is the reason why miRNAs can be potential targets for treatment, diagnosis and prognosis in ALL.

#### **3.2 The role of miRNAs in ALL development**

Besides the involvement of aberrant miRNAs expression in malignant lymphopoiesis, it may be related to drug resistance mechanisms. Focusing on promoting the reversal expression of such miRNA may enhance disease management and improve clinical outcomes. By comparing miRNAs expression in both normal pediatric bone marrow and pediatric ALL, an overexpression of miR-100, miR-196b in ALL and lower expression of let-7e in normal bone marrow were reported [37]. In a study conducted in childhood B-ALL and T-ALL, the miRNA expression profile in the bone marrow was evaluated to discriminate between T-ALL and B-ALL. The results indicated a downregulation of miR-708-5p, miR-497-5p, miR-151a-5p, miR-151b, miR-371b-5p, miR-455-5p, miR-195-5p, miR-1266-5p, miR-574-5p, miR-425-5p and upregulation of miR-450b-5p, miR-450a-5p, miR-542-5p, miR-424-5p, miR-629-5p, miR-29c-5p in T-ALL. Further machine process analyses showed that miR-29c-5p, involved in calcium signaling, is involved in B-cell fate and it is the best discriminator to stablish childhood B-ALL or T-ALL. In addition, clinical samples of T-ALL were studied and reveled that miR-223, miR-19b, miR-20a, miR-92, miR-142-3p, miR-150, miR-93, miR-26a, miR-16, and miR-342., miR-19b,-20a,-26a,-92 and miR-223 target T-ALL tumor suppressors, such as IKAROS, PTEN, BIM, PHF6, NF1 and FBXW7 [37], which means that overexpression of such miRNAs could lead to ALL development and represents a potential therapeutic target.

The identification of miRNAs that target genes of MYC, TFs of CDX2 and IncRNA of XIST could represent an approach to ALL therapeutics, due to that the aforementioned genes may play important role in the development of B-ALL. NOTCH1 is another important target due to mutations of gain of function, which are implicated in T-ALL development as well as NOTCH1 and NOTCH2 that take part in B-malignances [38]. **Table 1** exhibits a list of miRNAs involved in ALL development and pathogenesis considered as potential therapeutic targets.
