**4. Alternative splicing**

This post-transcriptional process is one of the key steps of messenger RNA maturation (mRNA) but apart from that, it allows to elevate complexity of the cellular proteome. A core complex called spliceosome is responsible for excision of introns from pre-mRNA in the nucleus. It consists of five small nuclear ribonucleoproteins (snRNPs) – U1, U2, U5, U4/U6 – and small nuclear RNA (snRNA). Splicing is regulated by *cis*-acting elements, which are the nucleotide sequences of primary transcript, *trans*-acting elements, which are the splicing factors or other RNA binding proteins regulating splicing: heterogenous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. The nucleotide sequence of the transcript promotes or represses splicing at the certain sites. The *trans*-acting elements can antagonize the activity of splicing factors leading to change in splice site selection, known as alternative splicing event. For an overview of the splicing process or interactions of RNA binding proteins with regulators of alternative splicing see: [49–51]. Furthermore, modification of RNA within the *cis*-acting region (like m6A) is one

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control the transcript fate and expression of a particular protein.

the biological significance of this has not been studied [55].

marker in therapy involving sulfonamid drugs [62, 63].

of the major factors that has impact on the binding of *trans*-acting elements (as described above). It has been found that the m6A modification enhances binding by RBPs such as hnRNP A2B1 [52] or SRSF3, whilst respells SRSF10 from transcript [53], exerting the effect on splicing of mRNA. This demonstrates how two distinct post-transcriptional events, RNA methylation and splicing, can cross-talk to tightly

Extracellular signals lead to modification of operation of *trans*-acting proteins, affecting or enhancing their action. Therefore, activity of regulatory elements involved in this process is sensitive to environmental signals such as cytokines or hypoxia. Through modulation of alternative splicing, the level of proteins' isoforms can be adjusted according to the circumstances. Additionally to selective intron skipping/excision, alternative splicing can affect length of poly(A) region. As a result, this influence stability of the mRNA, causing its enhanced decay or translation. Apart from that, mutation of the gene sequence modifies binding of RBPs what has impact on the ultimate expression level. Some of the splicing regulators were reported to play an oncogenic role, whilst others act as tumor suppressors. Apoptosis-stimulating Protein of TP53–2 (ASPP2) is a tumor suppressor enhancing TP53-mediated apoptosis. It has been found that a splice variant ASPP2κ is overexpressed in AML and displays anti-apoptotic function, therefore supporting

Analysis of the transcriptome profile revealed that progression of CML from chronic phase (CML-CP) towards acute blastic phase (CML-BP) is accompanied by changes in splicing pattern of genes, thus affecting spliceosome. The exon skipping event in hnRNP A1 led to accumulation of bigger isoform in the CML-BP, though

Altogether, it makes the alternative splicing machinery a powerful tool for cancer cells to support their survival. On the other hand, the necessity to achieve certain modifications through splicing makes cancer cells dependent on this pro-

Activity of SR proteins can be modulated by modifications: phosphorylation of SR proteins can affect activity and switch general splicing repressor to selective activator [56], methylation of SRSF1 by the protein arginine methyltransferase 5 (PRMT5) modulates splicing of many proteins involved in proliferation, therefore supports leukemia development [57]. Its overexpression exerts oncogenic effect increasing aggressiveness of leukemia cells [58]. Specific inhibitors of PRMT5 have been clinically tested for potential treatment of blood and solid cancers [59]. As transcription of PRMT5 it directly stimulated by MYC transcription factor [60], thus leukemia cells overexpressing MYC could be selected for treatment with

A screen of RBPs playing role in AML revealed that RNA binding motif protein 39 (RBM39) plays a significant role in RNA splicing and stimulates proliferation of leukemia cells [61]. A sulfonamid drugs (indisulam, tasisulam, E7820 and CQS,), which are an example of proteolysis-targeting chimeras (PROTACs) compounds, led to polyubiquitination and proteasomal degradation of RBM39, what exerts anticancer activity [62]. The effect depended on the CUL4-DCAF15 E3 ubiquitin ligase, therefore the level of expression of this enzyme could be used as indicative

Leukemia development and progression can be triggered by occurrence of point mutations in splicing factors such as SF3B. Activity of spliceosomal complex containing the mutated protein, but not wild type SF3B, can be blocked by a specific small molecule inhibitor H3B-8800 [64]. It has been shown that AML cells with mutated U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1), a component of spliceosome, display increased sensitivity to sudemycin – a drug targeting SF3BP1, both

cess. Such regulation creates an opportunity for therapeutic targeting.

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

proliferation of the cells [54].

PRMT5 inhibitors.

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

of the major factors that has impact on the binding of *trans*-acting elements (as described above). It has been found that the m6A modification enhances binding by RBPs such as hnRNP A2B1 [52] or SRSF3, whilst respells SRSF10 from transcript [53], exerting the effect on splicing of mRNA. This demonstrates how two distinct post-transcriptional events, RNA methylation and splicing, can cross-talk to tightly control the transcript fate and expression of a particular protein.

Extracellular signals lead to modification of operation of *trans*-acting proteins, affecting or enhancing their action. Therefore, activity of regulatory elements involved in this process is sensitive to environmental signals such as cytokines or hypoxia. Through modulation of alternative splicing, the level of proteins' isoforms can be adjusted according to the circumstances. Additionally to selective intron skipping/excision, alternative splicing can affect length of poly(A) region. As a result, this influence stability of the mRNA, causing its enhanced decay or translation. Apart from that, mutation of the gene sequence modifies binding of RBPs what has impact on the ultimate expression level. Some of the splicing regulators were reported to play an oncogenic role, whilst others act as tumor suppressors.

Apoptosis-stimulating Protein of TP53–2 (ASPP2) is a tumor suppressor enhancing TP53-mediated apoptosis. It has been found that a splice variant ASPP2κ is overexpressed in AML and displays anti-apoptotic function, therefore supporting proliferation of the cells [54].

Analysis of the transcriptome profile revealed that progression of CML from chronic phase (CML-CP) towards acute blastic phase (CML-BP) is accompanied by changes in splicing pattern of genes, thus affecting spliceosome. The exon skipping event in hnRNP A1 led to accumulation of bigger isoform in the CML-BP, though the biological significance of this has not been studied [55].

Altogether, it makes the alternative splicing machinery a powerful tool for cancer cells to support their survival. On the other hand, the necessity to achieve certain modifications through splicing makes cancer cells dependent on this process. Such regulation creates an opportunity for therapeutic targeting.

Activity of SR proteins can be modulated by modifications: phosphorylation of SR proteins can affect activity and switch general splicing repressor to selective activator [56], methylation of SRSF1 by the protein arginine methyltransferase 5 (PRMT5) modulates splicing of many proteins involved in proliferation, therefore supports leukemia development [57]. Its overexpression exerts oncogenic effect increasing aggressiveness of leukemia cells [58]. Specific inhibitors of PRMT5 have been clinically tested for potential treatment of blood and solid cancers [59]. As transcription of PRMT5 it directly stimulated by MYC transcription factor [60], thus leukemia cells overexpressing MYC could be selected for treatment with PRMT5 inhibitors.

A screen of RBPs playing role in AML revealed that RNA binding motif protein 39 (RBM39) plays a significant role in RNA splicing and stimulates proliferation of leukemia cells [61]. A sulfonamid drugs (indisulam, tasisulam, E7820 and CQS,), which are an example of proteolysis-targeting chimeras (PROTACs) compounds, led to polyubiquitination and proteasomal degradation of RBM39, what exerts anticancer activity [62]. The effect depended on the CUL4-DCAF15 E3 ubiquitin ligase, therefore the level of expression of this enzyme could be used as indicative marker in therapy involving sulfonamid drugs [62, 63].

Leukemia development and progression can be triggered by occurrence of point mutations in splicing factors such as SF3B. Activity of spliceosomal complex containing the mutated protein, but not wild type SF3B, can be blocked by a specific small molecule inhibitor H3B-8800 [64]. It has been shown that AML cells with mutated U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1), a component of spliceosome, display increased sensitivity to sudemycin – a drug targeting SF3BP1, both

*Acute Leukemias*

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

HuR is a good candidate for cancer treatment strategy.

the C/EBP1α protein synthesis.

**4. Alternative splicing**

possibility to treat acute leukemia patients.

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

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

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

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.

This post-transcriptional process is one of the key steps of messenger RNA maturation (mRNA) but apart from that, it allows to elevate complexity of the cellular proteome. A core complex called spliceosome is responsible for excision of introns from pre-mRNA in the nucleus. It consists of five small nuclear ribonucleoproteins (snRNPs) – U1, U2, U5, U4/U6 – and small nuclear RNA (snRNA). Splicing is regulated by *cis*-acting elements, which are the nucleotide sequences of primary transcript, *trans*-acting elements, which are the splicing factors or other RNA binding proteins regulating splicing: heterogenous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. The nucleotide sequence of the transcript promotes or represses splicing at the certain sites. The *trans*-acting elements can antagonize the activity of splicing factors leading to change in splice site selection, known as alternative splicing event. For an overview of the splicing process or interactions of RNA binding proteins with regulators of alternative splicing see: [49–51]. Furthermore, modification of RNA within the *cis*-acting region (like m6A) is one

**270**

*in vitro* and *in vivo*. Transcripts altered by sudemycin treatment encoded proteins involved in receptor and signal transduction activities [65]. Noteworthy, SF3B1 has distinct effect on the transcript splicing than SRSF2. Mutation in any of them led to hyperactivation of NF-κB signaling, whilst simultaneous mutation of both SF3B1 and SRSF2 displayed synthetically lethal effect [66].

The splicing regulatory network is complex and full of various cross-talk regulations and interactions. Due to interactive nature of regulatory factors, which influence each other's activity, lowering the level of one factor activates a compensatory mechanism that is mediated by another factor. This refers to both SR [67] as well as hnRNP proteins [68]. Thus, the results should be interpreted cautiously, because the observed effect resulting from a loss of one splicing factor may in fact be the secondary effect of a change in the network. Nevertheless, targeting of the *cis*- or *trans*-regulating elements gives possibility to hit precisely the source of oncogenic transformation.

#### **5. Translation initiation**

Previously we described the processes, which regulate translation of the specific RNA in a controlled manner. The initiation of translation is another step on the way of protein synthesis, which is tightly controlled. The process can be remodeled by multiple cellular intrinsic signaling pathways that can be active in malignant cells.

Constitutive activation of the PI3K/Akt/mTOR signaling pathway [69, 70] has been observed in various types of leukemia, including acute lymphoblastic leukemia (ALL), Philadelphia (Ph) chromosome positive and Ph-like acute lymphoblastic leukemia (BCR-ABL1-like ALL) or AML. Continuous activity of the PI3K/Akt/ mTOR pathway contributes to unregulated proliferation and leads to resistance to therapy with tyrosine kinase inhibitors (TKI) [71]. Activation of mTOR results in phosphorylation of S6K kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP1), promoting cap-dependent mRNA initiation of translation and increased protein synthesis in leukemia cells [72]. On the other hand, mTOR pathway stimulates cap-independent translation mediated by internal ribosome entry sites IRES, mainly by activation of eIF4A helicase [73]. Another signaling pathway that regulates initiation of translation is the Ras/MAPK/ERK pathway. Its activity has also been found in leukemia cells [71, 74]. Activation of that pathway resulted in phosphorylation of the translation initiation factor eIF4E by MNK1/2 kinases. This contributed to increased β-catenin mRNA translation efficiency and activation of the Wnt/β-catenin signaling pathway, which plays an important role in differentiation and proliferation of leukemia cells [71, 75]. Microenvironmental signals, such as acute hypoxia or nutrient deprivation, trigger so called Integrated Stress Response (ISR) pathway, which shapes the mRNA translation. There are four protein kinases activated dependently on the stressor type: GCN – amino acid deprivation, PKR – appearance of viral RNA, PERK – accumulation of unfolded/ misfolded proteins in the ER and HRI – oxidative stress, heme deficiency, osmotic shock and heat shock. Activation of these kinases in response to stress leads to phosphorylation of eukaryotic initiation factor 2 subunit alpha (eIF2α) and 4E-BP, which orchestrate number of downstream events regulating translation. ISR has been shown to be active in leukemia cells and displayed pro-survival properties of those cells [76].

Changes in cellular signaling provide a great opportunity for the anti-leukemia treatment strategy. One is based on the inhibition of PI3K/Akt/mTOR [77] and Ras/MAPK/ERK [78] signaling pathways. Rapamycin, an inhibitor of the mTOR signaling pathway, has been tested in the context of leukemia treatment [79].

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

Moreover, PP242 and hippuristanol, inhibitors of mTOR-eIF4A pathways, also have the potential to overcome TKI resistance [73]. Recent reports have shown that ribavirin, which is used as an antiviral drug, inhibits the mTOR/eIF4E and ERK/Mnk1/ eIF4E pathways in leukemia cells expressing BCR-ABL1 oncogene and ultimately leads to reduction of anti-apoptotic proteins, inhibition of proliferation and consequently apoptosis of leukemia cells [80]. Salubrinal, guanabenz and Sephin1 are known agents inhibiting activity of phosphatase, which dephosphorylates eIF2α. Their effectiveness has been studied mainly in the context of neurological disorders, but leukemia should also be included into this research. Inactivation of ISR pathway can constitute a significant treatment strategy. The best described group of molecules targeting ISR are inhibitors of the kinases phosphorylating eIF2α, i.e. PERK kinase. The compounds GSK2606414 and GSK2656157 were designed to bind selectively to the ATP binding pocket of the PERK kinase and to inhibit PERK activity. Their potency has been studied in a vast number of cancers. Unfortunately, the use of those inhibitors caused serious side effects [81] and recently not specific effects have been reported [82]. Alternatively, a small molecule ISRIB (ISR inhibitor) is another example of the drug that inhibits general translation. It acts downstream of the eIF2α factor in the ISR signaling pathway and through direct interaction with eIF2B abolishes the phosphorylation effect of eIF2α [83].

Initiation of the translation process can be altered by aberrant cell signaling leading to enhanced expression of proteins, which play a pro-survival role and support cell proliferation. Therefore, interference with elements enabling the stimulated translation, could be one of the strategies that, by targeting general protein synthesis, restrict propagation potential of acute leukemia cells. The broad spectrum of proteins which synthesis might be disturbed by such treatment, has both advantages and disadvantages. On one hand such treatment will affect most of actively translating cells in the body. On the other hand however, this attitude represents a powerful tool to block highly proliferative acute leukemia cells by limiting their ability to propagate. Moreover, taking into account the heterogeneity of cancer, it might be effective towards broader spectrum of leukemia cell clones and push the clonal selection towards less proliferative, so less aggressive form of cancer. This in turn enables other drugs, which can be then used in combinatory treatment

Machinery that physically executes the protein synthesis on the matrices of mRNA is based on ribosomes. This complex entities are formulated of rRNA core and ribosomal proteins (RPs) of small (RPS) or large (RPL) subunit. Recent evidence demonstrated that ribosomes can contain different RPL/RPS, thus indicating a heterogeneity among ribosomes. Additionally, different expression of some RPL and RPS in the tissues has been observed. There are RPs, such as RPL38, which regulate translation of the Homeobox genes during embryo development [84], showing involvement of RPs in directing tissue organization. This heterogeneity of the translational machinery is further amplified by proteins associating/interacting

Existence of ribosomopathies demonstrates that RPs can play significant role in determining the cell fate. The first ribosomopathy which has been recognized was the Diamond-Blackfan anemia (DBA) caused by defect in RPS19 gene, what leads to the bone marrow failure [88]. Since then, more similar aberrations related to pathological state have been discovered. It has been observed that ribosomopathies are often related to cancers including leukemia, bone marrow failure and anemia.

with ribosomes [85, 86] (reviewed in more detail here [87]).

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

to exert the beneficial effect.

**6. Ribosomal proteins**

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

Moreover, PP242 and hippuristanol, inhibitors of mTOR-eIF4A pathways, also have the potential to overcome TKI resistance [73]. Recent reports have shown that ribavirin, which is used as an antiviral drug, inhibits the mTOR/eIF4E and ERK/Mnk1/ eIF4E pathways in leukemia cells expressing BCR-ABL1 oncogene and ultimately leads to reduction of anti-apoptotic proteins, inhibition of proliferation and consequently apoptosis of leukemia cells [80]. Salubrinal, guanabenz and Sephin1 are known agents inhibiting activity of phosphatase, which dephosphorylates eIF2α. Their effectiveness has been studied mainly in the context of neurological disorders, but leukemia should also be included into this research. Inactivation of ISR pathway can constitute a significant treatment strategy. The best described group of molecules targeting ISR are inhibitors of the kinases phosphorylating eIF2α, i.e. PERK kinase. The compounds GSK2606414 and GSK2656157 were designed to bind selectively to the ATP binding pocket of the PERK kinase and to inhibit PERK activity. Their potency has been studied in a vast number of cancers. Unfortunately, the use of those inhibitors caused serious side effects [81] and recently not specific effects have been reported [82]. Alternatively, a small molecule ISRIB (ISR inhibitor) is another example of the drug that inhibits general translation. It acts downstream of the eIF2α factor in the ISR signaling pathway and through direct interaction with eIF2B abolishes the phosphorylation effect of eIF2α [83].

Initiation of the translation process can be altered by aberrant cell signaling leading to enhanced expression of proteins, which play a pro-survival role and support cell proliferation. Therefore, interference with elements enabling the stimulated translation, could be one of the strategies that, by targeting general protein synthesis, restrict propagation potential of acute leukemia cells. The broad spectrum of proteins which synthesis might be disturbed by such treatment, has both advantages and disadvantages. On one hand such treatment will affect most of actively translating cells in the body. On the other hand however, this attitude represents a powerful tool to block highly proliferative acute leukemia cells by limiting their ability to propagate. Moreover, taking into account the heterogeneity of cancer, it might be effective towards broader spectrum of leukemia cell clones and push the clonal selection towards less proliferative, so less aggressive form of cancer. This in turn enables other drugs, which can be then used in combinatory treatment to exert the beneficial effect.
