**2. Modifications of RNA**

Posttranscriptional modifications have been found in non-coding RNAs such as ribosomal RNAs (rRNA) and transfer RNAs (tRNA) as well as in messenger RNAs (mRNA). There are about 150 modifications discovered by far. They include pseudouridylation (ψ), methylation or deamination of adenosine to inosine (A-to-I editing). Such modifications have impact on the splicing and translation of mRNA and contribute to epitranscriptional regulation of gene expression. Some modifications exist only in the coding sequence (like A-to-I editing), whilst others are deposited only in a 5′-untranslated regions (5'UTR) such as 5-methylcytosine (m5C) and 7-methylguanosine (m7G). The N6-methyladenosine (m6A) modification is ubiquitously present and deposited in along the mRNA coding sequence and 5′/3′UTRs.

The m6A modification is added to the mRNA in the nucleus by so called 'writers' or removed by 'erasers' and is recognized by proteins which bind to m6A methylated mRNA (so called 'readers'). It has impact on the mRNA stability, export from the nucleus, decay and translation (for recent review on the role of RNA modifications in cancer, including acute myelogenous leukemia (AML) see: [6–8]). The m6A modification has been found to play a critical role in AML development and progression (for review see: [9, 10]).

The m6A writer proteins - methyltransferase-like protein (METTL) 3 and 14 are overexpressed in AML. It was reported that their deletion limits the cancerogenic cellular potential [11, 12]. On the other hand, METTL3 overexpression stimulated the translation of Myc, Bcl-2 and PTEN what contributed to increased proliferation and survival of AML cells [13]. Controversially, increased expression of fat mass and obesity-associated demethylase (FTO), which acts as a m6A eraser, also led to higher level of oncogene expression. Moreover, its inhibition reduced growth of AML cancer cells [14]. Activity of FTO has been found to be directly inhibited by R-2-hydroxyglutarate (R-2HG) leading to loss of stability of Myc mRNA and decreased proliferation rate of leukemic cells [15]. This effect is postulated to result from discrepancy of mRNA triage for translation or decay of pro- and anti-oncogenic proteins in respect to the presence of m6A deposition in mRNA [8].

Though YTHDF2, the m6A reader, appears not to be required for normal hematopoietic stem cells, it occurs to be essential for AML cells similarly to METTL3 and FTO. Its overexpression facilitates AML cells propagation, whereas its silencing disables proliferative and clonogenic potential of leukemia cells. Thus, YTHDF2 seems to be a good therapeutic target in AML, which would enable the selective eradication of cancer cells whilst spearing healthy hematopoietic stem cells [16].

Another m6A reader proteins might also play a key role in the regulation of cancer development. The insulin-like growth factor 2 mRNA-binding protein (IF2BP1–3) stabilizes m6A-modified mRNAs such as *MYC* oncogene, thus enhancing its translation and contributing to oncogenesis [17].

Apart from modification of mRNA, also pseudouridylation of tRNA contributes to AML progression. The tRNAs that contain 5′ terminal oligoguanine (TOG)

**269**

*Targeting of Post-Transcriptional Regulation as Treatment Strategy in Acute Leukemia*

are the source of 18 nucleotide regulatory sequences (mTOGs), which stimulate differentiation and limit proliferation of hematopoietic stem cells (HSC) by inhibiting translation initiation in HSC. This effect depends on the presence of ψ on the mTOGs. It has been found that development of AML is accompanied by decreased level of pseudouridine synthase 7 (PUS7). Downregulation of PUS7 abolished healthy stem cells differentiation and increased translation demonstrating significance of ψ modification in the development of AML [18]. The oncogenic mTOGs, if attenuated by specific inhibitors, could constitute an effective therapeutic target. The above examples show that the post-transcriptional regulation of gene expression at the step of RNA modifications constitutes a potent target to disable expression of some oncogenes that should allow to switch the cell fate back towards

Activation of RNA binding proteins (RBPs) constitutes an additional layer of posttranscriptional regulation, which has a great impact on the final protein level in the cell. The main function of RNA binding proteins is to recognize the primary transcript (pre-mRNA) and assemble ribonucleoprotein complexes, what governs processes of pre-mRNA maturation i.e. splicing, polyadenylation, attachment of a guanyl cap at the 5′ end of pre-mRNA and RNA modifications. Moreover, RBPs binding to the target mRNA is required for proper mRNA transportation from the nucleus to the cytoplasm and distribution into various cellular compartments. Additionally, *trans*-acting regulatory RNA binding proteins have the ability to affect translation of the specific mRNA, mainly through the interaction with untranslated regions (3′UTR and 5′UTR) and coding region of mature mRNA, what results in

Considering the multifunctional properties of RNA binding proteins, any alterations in those proteins' activity are associated with multiple cancers (reviewed in [19]), including leukemias (reviewed in [20]), and provide a substantial therapeu-

The Musashi (MSI) RNA binding proteins (MSI1 and MSI2) contribute to development of various types of cancer. Their elevated expression has been demonstrated in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia blastic phase (CML-BP) [21–23]. MSI proteins regulate translation of mRNAs encoding proteins involved in several oncogenic signaling pathways, such as MYC [24], TGFβ/SMAD3 [25] and PTEN/ mTOR [26]. Thus, inhibition of MSI RNA-binding activity could demonstrate a novel therapeutic strategy, probably not only in solid tumors but in hematological malignancies as well. A small molecule Ro 08–2750 (Ro) has been shown recently to bind selectively to MSI2 and interfere with its mRNA binding activity, thus triggering increased apoptosis and inhibition of known MSI targets in myeloid leukemia cells [27]. Other agents with presumptive MSI1 inhibitory activity have also been tested and they include (−)-gossypol (natural phenol extracted from cottonseed) [28] and ω − 9 monounsaturated fatty acids (e.g. oleic acid) [29]. Although those agents display inhibitory effects on MSI1 activity, the specificity of both should be

Another RNA binding protein involved in mRNA translation and deregulated in leukemia is DDX3. Mutations in DDX3 gene display oncogenic potential in T-cell lymphoma [30] and lymphocytic leukemia [31]. A small molecule inhibitor

(RK-33) targeting DDX3, which has been tested so far, demonstrates the pro-apoptotic activity. Its administration promoted higher sensitivity to radiation in lung cancer

*DOI: http://dx.doi.org/10.5772/intechopen.94421*

appropriate hematopoietic differentiation.

**3. RNA binding proteins**

changes in ribosome recruitment.

tic opportunity.

further confirmed.

#### *Targeting of Post-Transcriptional Regulation as Treatment Strategy in Acute Leukemia DOI: http://dx.doi.org/10.5772/intechopen.94421*

are the source of 18 nucleotide regulatory sequences (mTOGs), which stimulate differentiation and limit proliferation of hematopoietic stem cells (HSC) by inhibiting translation initiation in HSC. This effect depends on the presence of ψ on the mTOGs. It has been found that development of AML is accompanied by decreased level of pseudouridine synthase 7 (PUS7). Downregulation of PUS7 abolished healthy stem cells differentiation and increased translation demonstrating significance of ψ modification in the development of AML [18]. The oncogenic mTOGs, if attenuated by specific inhibitors, could constitute an effective therapeutic target.

The above examples show that the post-transcriptional regulation of gene expression at the step of RNA modifications constitutes a potent target to disable expression of some oncogenes that should allow to switch the cell fate back towards appropriate hematopoietic differentiation.

## **3. RNA binding proteins**

*Acute Leukemias*

5′/3′UTRs.

**2. Modifications of RNA**

progression (for review see: [9, 10]).

(specific mRNA sequences such as internal ribosome entry sites IRES, mRNA posttranscriptional modifications) and *trans*-acting factors (such as RNA-binding proteins, microRNAs) that bind to mRNA [5]. Furthermore, changes in the cellular signaling can also trigger translational reprogramming. Based on the significant role that all steps of the translation regulation play in development of cancers, including hematological malignancies, and their pro-survival and adaptive function, thera-

Posttranscriptional modifications have been found in non-coding RNAs such as ribosomal RNAs (rRNA) and transfer RNAs (tRNA) as well as in messenger RNAs (mRNA). There are about 150 modifications discovered by far. They include pseudouridylation (ψ), methylation or deamination of adenosine to inosine (A-to-I editing). Such modifications have impact on the splicing and translation of mRNA and contribute to epitranscriptional regulation of gene expression. Some modifications exist only in the coding sequence (like A-to-I editing), whilst others are deposited only in a 5′-untranslated regions (5'UTR) such as 5-methylcytosine (m5C) and 7-methylguanosine (m7G). The N6-methyladenosine (m6A) modification is ubiquitously present and deposited in along the mRNA coding sequence and

The m6A modification is added to the mRNA in the nucleus by so called 'writers' or removed by 'erasers' and is recognized by proteins which bind to m6A methylated mRNA (so called 'readers'). It has impact on the mRNA stability, export from the nucleus, decay and translation (for recent review on the role of RNA modifications in cancer, including acute myelogenous leukemia (AML) see: [6–8]). The m6A modification has been found to play a critical role in AML development and

The m6A writer proteins - methyltransferase-like protein (METTL) 3 and 14 are overexpressed in AML. It was reported that their deletion limits the cancerogenic cellular potential [11, 12]. On the other hand, METTL3 overexpression stimulated the translation of Myc, Bcl-2 and PTEN what contributed to increased proliferation and survival of AML cells [13]. Controversially, increased expression of fat mass and obesity-associated demethylase (FTO), which acts as a m6A eraser, also led to higher level of oncogene expression. Moreover, its inhibition reduced growth of AML cancer cells [14]. Activity of FTO has been found to be directly inhibited by R-2-hydroxyglutarate (R-2HG) leading to loss of stability of Myc mRNA and decreased proliferation rate of leukemic cells [15]. This effect is postulated to result from discrepancy of mRNA triage for translation or decay of pro- and anti-onco-

genic proteins in respect to the presence of m6A deposition in mRNA [8].

ing its translation and contributing to oncogenesis [17].

Though YTHDF2, the m6A reader, appears not to be required for normal hematopoietic stem cells, it occurs to be essential for AML cells similarly to METTL3 and FTO. Its overexpression facilitates AML cells propagation, whereas its silencing disables proliferative and clonogenic potential of leukemia cells. Thus, YTHDF2 seems to be a good therapeutic target in AML, which would enable the selective eradication of cancer cells whilst spearing healthy hematopoietic stem cells [16]. Another m6A reader proteins might also play a key role in the regulation of cancer development. The insulin-like growth factor 2 mRNA-binding protein (IF2BP1–3) stabilizes m6A-modified mRNAs such as *MYC* oncogene, thus enhanc-

Apart from modification of mRNA, also pseudouridylation of tRNA contributes

to AML progression. The tRNAs that contain 5′ terminal oligoguanine (TOG)

peutic targeting of those mechanisms has been proposed and studied.

**268**

Activation of RNA binding proteins (RBPs) constitutes an additional layer of posttranscriptional regulation, which has a great impact on the final protein level in the cell. The main function of RNA binding proteins is to recognize the primary transcript (pre-mRNA) and assemble ribonucleoprotein complexes, what governs processes of pre-mRNA maturation i.e. splicing, polyadenylation, attachment of a guanyl cap at the 5′ end of pre-mRNA and RNA modifications. Moreover, RBPs binding to the target mRNA is required for proper mRNA transportation from the nucleus to the cytoplasm and distribution into various cellular compartments. Additionally, *trans*-acting regulatory RNA binding proteins have the ability to affect translation of the specific mRNA, mainly through the interaction with untranslated regions (3′UTR and 5′UTR) and coding region of mature mRNA, what results in changes in ribosome recruitment.

Considering the multifunctional properties of RNA binding proteins, any alterations in those proteins' activity are associated with multiple cancers (reviewed in [19]), including leukemias (reviewed in [20]), and provide a substantial therapeutic opportunity.

The Musashi (MSI) RNA binding proteins (MSI1 and MSI2) contribute to development of various types of cancer. Their elevated expression has been demonstrated in acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia blastic phase (CML-BP) [21–23]. MSI proteins regulate translation of mRNAs encoding proteins involved in several oncogenic signaling pathways, such as MYC [24], TGFβ/SMAD3 [25] and PTEN/ mTOR [26]. Thus, inhibition of MSI RNA-binding activity could demonstrate a novel therapeutic strategy, probably not only in solid tumors but in hematological malignancies as well. A small molecule Ro 08–2750 (Ro) has been shown recently to bind selectively to MSI2 and interfere with its mRNA binding activity, thus triggering increased apoptosis and inhibition of known MSI targets in myeloid leukemia cells [27]. Other agents with presumptive MSI1 inhibitory activity have also been tested and they include (−)-gossypol (natural phenol extracted from cottonseed) [28] and ω − 9 monounsaturated fatty acids (e.g. oleic acid) [29]. Although those agents display inhibitory effects on MSI1 activity, the specificity of both should be further confirmed.

Another RNA binding protein involved in mRNA translation and deregulated in leukemia is DDX3. Mutations in DDX3 gene display oncogenic potential in T-cell lymphoma [30] and lymphocytic leukemia [31]. A small molecule inhibitor (RK-33) targeting DDX3, which has been tested so far, demonstrates the pro-apoptotic activity. Its administration promoted higher sensitivity to radiation in lung cancer

DDX3-overexpressing cells [32, 33], thus providing an argument to develop and improve DDX3 inhibitors, which can target cancer cells, including leukemia.

The activity of HuR RNA binding protein is also deregulated in some types of leukemia [34–37]. Elevated HuR level promotes tumorigenesis, thus targeting HuR could be a promising anti-cancer therapy. A few chemical compounds against HuR activity have been tested so far. MS-444 small molecule inhibitor interfered with HuR binding to target ARE-mRNAs and showed anti-tumor properties in various types of cancers [38–40]. Quercetin and b-40 have been found to inhibit HuR binding to TNFα mRNA, what resulted in TNFα destabilization and decreased TNFα secretion [41]. A coumarin-derived and HuR-targeted small molecule inhibitor CMLD-2, exhibited cytotoxicity towards human lung cancer cells [42], proving that HuR is a good candidate for cancer treatment strategy.

Aberrations of other RNA binding proteins have been linked to the activity of BCR-ABL1, an oncoprotein responsible for chronic myeloid leukemia (CML) development. BCR-ABL1-dependent decrease of CUGBP1 level resulted in repression of the C/EBPβ mRNA translation [43]. As C/EBPβ transcriptional activity controls the maturation of hematopoietic cells in the myeloid lineage, its deficiency contributes to differentiation arrest of CML cells and CML progression to the blast crisis [44]. An increased level and activity of RNA binding proteins: hnRNP K [45], hnRNP A1 [46], hnRNP E2 [46], TLS/FUS [47] and La/SSB [48] have also been observed. These proteins regulate translation of important cancer-related factors: the hnRNP K protein positively regulates c-MYC mRNA translation, protein La/SSB promotes MDM2 mRNA translation, and increased hnRNP E2 activity leads to inhibition of the C/EBP1α protein synthesis.

Activities of RNA binding proteins described above result in the differentiation arrest of CML cell, but also their increased proliferation and survival. Considering the mentioned features, RNA binding proteins provide a significant therapeutic possibility to treat acute leukemia patients.

A single RBP interacts with a number of different mRNAs, and prerequisite for this is a presence of the RBP's binding sequence. The recognition motif for a given protein is often present in mRNAs encoding proteins needed in a certain process. For instance, mRNAs of cell cycle regulating proteins are bound by HuR. Thus, targeting activity of the specific RBP, interfering with its binding ability or masking the targeted sequence would impact the fate of a group of mRNAs. Therefore, this constitutes an opportunity to modulate synthesis of functionally related proteins.
