**4. Structural features of the different BCR-ABL protein variants**

Both c-ABL and BCR are rather large proteins with molecular sizes ranging from 145 to 160 kDa, respectively, and harboring numerous well defined structural conserved domains. The cABL is a non-receptor tyrosine kinase harboring several motifs that are required for its own enzymatic activity and to signal to other molecules. Intramolecular interactions occurring between the SH3 domain and the linker peptide connecting the SH2 and the tyrosine kinase domain, alongside with that occurring between the kinase and the SH2 domain, keep c-ABL in a close inactive state [23]. The central part of the protein is characterized by proline-rich (PxxP) stretches acting as docking sites for SH3 containing proteins and a DNA binding domain (DBD). Eventually the carboxy-terminal region contains an actin binding domain (ABD) which allows the interaction either with the monomeric- (G) and with the filamentous- (F) actin. Within cells, the ABL is distributed either in the nucleus and, to a lesser extent, in the cytoplasm where it plays distinct roles. The shuttling between the two compartments it steered by its nuclear-localization and nuclearexport signals and it is depending on different extracellular cues (*e.g.* cell to substratum adherence) [24]. While cytoplasmic c-ABL regulates several actin-dependent cellular processes, for example by positively controlling the filopodia exploration and the membrane ruffling [25], the nuclear c-ABL is a pivotal proapoptotic play-actor, playing a role in the cellular response to genotoxic stress (*e.g.* ionizing radiation) [26].

Alike c-ABL, BCR is a multidomains protein with a peculiarity consisting of a Dbl homology (DH) and a Rho-GAP domain that are localized in the central region and at the C-terminus of the protein, respectively. These domains act as Guanine Exchange Factor (GEF) and GTPase Activating regulatory elements (GAP) for some members of the Rho superfamily, including Cdc42, Rac1, Rac2 and RhoA. Additionally, BCR protein harbors other structural regions, including two lipid binding domains namely Pleckstrin Homology (PH) and Calcium-dependent lipidbinding domain (C2), which is localized in the central part of the protein, and an N-terminal 63 aminoacids long coiled-coil oligomerization peptide that is followed by a Serine/Threonine kinase domain. BCR expression is rather ubiquitous and enriched in brain. Differently from c-ABL, the subcellular localization of BCR is predominantly restricted to the cytoplasmic compartment [27].

The structural composition of BCR-ABL proteins may vary quite a lot depending from which fusion gene breakpoint one refer to (**Figure 1**).

However, the variation is always restricted to the BCR part, while the c-ABL part remains constant in all the different transcript variants. This is in itself an indication that c-ABL is mostly responsible for its transforming properties. Briefly, all BCR-ABL proteins share the same c-ABL part with all the prominent structural

**235**

**leukemogenic properties**

*The Paradigm of Targeting an Oncogenic Tyrosine Kinase: Lesson from BCR-ABL*

features of c-ABL, including SH3, SH2, tyrosine kinase, proline rich regions, DNAbinding and Actin Binding Domains. By contrast, the distinguishing part is represented by the BCR peptide. In the shortest BCR-ABL variant (p190) or alternatively named p185, the BCR portion encodes for a peptide of approximately 490 aminoacids encoded by the first BCR exon, encompassing the very N-terminal coiled-coil oligomerization domain and the serine/threonine kinase domain, fused to the ABL. Conversely, in the longest BCR-ABL variant, p230, the BCR portion harbors all BCR structural domains with the exception of the GAP that is truncated. Eventually, the most common BCR-ABL variant, p210, encodes for a chimeric protein in which the BCR portion comprises the coiled-coil, Ser/Thr, Rho-GEF and PH domains. Crucial for the constitutive activation of the c-ABL tyrosine kinase is the BCR oligomerization domain that promotes either the dimerization or tetramerization of the protein [28]. In this way the BCR-ABL proteins can cross-phosphorylate each other on tyrosine residues in their kinase-activation loops. BCR-ABL phosphorylated tyrosine residues usurp the physiological functions of the normal ABL and can act as docking sites for SH2-domain containing proteins that contribute in activating downstream signaling pathways. On the whole this leads to clear readouts comprising deregulated cellular proliferation, decreased adherence of leukemia cells to the bone marrow stroma and reduced apoptotic response to mutagenic stimuli. Alike the BCR, but differently from the c-ABL protein, strikingly all the BCR-ABL chimeric proteins display a cytoplasmic localization, though all retain both the nuclear-localization and nuclear-export peptide sequences. The main reason for its cytoplasmic localization is its constitutively activated tyrosine kinase activity that thus allows to the chimeric tyrosine kinase to interact and cross talk with a number of proteins, thus exerting its leukemogenic effect. Interestingly, upon BCR-ABL pharmacological inactivation (*i.e.* Imatinib) and concurrent blocking of its nuclear export (*i.e.* leptomycin B) the protein re-localizes within the nuclear compartment and it is trapped there. Astonishingly, upon Imatinib removal and the tyrosine kinase activity of the nuclear BCR-ABL is reactivated it is converted from an

*Structural features of the different Bcr-Abl protein variants. Linear depiction of the functional motif composition of the different BCR-ABL proteins: p190, p210 and p230. CC: Coiled-coil; S/T kinase: Serine/ threonine kinase; DH: Dbl-homology; PH: Pleckstrin homology; C2: Ca2+-dependent membrane-targeting module; Rac-GAP: Rac GTPase; SH3: Src Homology3; SH2: Src Homology2; Tyr kinase: Tyrosine kinase: DBD: DNA binding domain; ABD: Actin binding domain; PXXP: Proline rich region, where X indicates any* 

antiapoptotic to proapoptotic protein thus inducing cell death [29].

**5. "In-vitro" and "in-vivo" tools for the assessment of the BCR-ABL** 

The importance of BCR-ABL in leukemogenesis/neoplastic transformation has been examined in numerous "*in-vitro*" and "*in-vivo*" biological systems, including

*DOI: http://dx.doi.org/10.5772/intechopen.97528*

**Figure 1.**

*aminoacid.*

*The Paradigm of Targeting an Oncogenic Tyrosine Kinase: Lesson from BCR-ABL DOI: http://dx.doi.org/10.5772/intechopen.97528*

#### **Figure 1.**

*Advances in Precision Medicine Oncology*

fused to multiple tyrosine kinases encoding genes, other than ABL, including Fibroblast Growth Factor Receptor1 (FGFR1) -t(8;22)- [15, 16], Platelet Derived Growth Factor Receptor A (PDGFRA) -t(4;22)- [17, 18], RET -t(10;22)- [19] and Jak2 -t(9;22)-[20–22] producing different fusion transcripts that are all encoding for cytoplasmic chimeric proteins displaying dysregulated tyrosine kinase enzymatic activity and onocogenic properties. The causal reason behind the commonality of BCR as fusion partner is not well understood. As we have previously discussed it has been speculated that genes such as BCR are located near chromosomal fragile sites that show breaks or gaps on metaphase chromosomes due to replication stress which are prone to breakage and translocation as result. Interestingly, though BCR fusion genes have also been detected in solid tumors, to date BCR fusion proteins that behave as cancer drivers have solely been identified in hematological cancers.

**4. Structural features of the different BCR-ABL protein variants**

predominantly restricted to the cytoplasmic compartment [27].

from which fusion gene breakpoint one refer to (**Figure 1**).

The structural composition of BCR-ABL proteins may vary quite a lot depending

However, the variation is always restricted to the BCR part, while the c-ABL part remains constant in all the different transcript variants. This is in itself an indication that c-ABL is mostly responsible for its transforming properties. Briefly, all BCR-ABL proteins share the same c-ABL part with all the prominent structural

Both c-ABL and BCR are rather large proteins with molecular sizes ranging from 145 to 160 kDa, respectively, and harboring numerous well defined structural conserved domains. The cABL is a non-receptor tyrosine kinase harboring several motifs that are required for its own enzymatic activity and to signal to other molecules. Intramolecular interactions occurring between the SH3 domain and the linker peptide connecting the SH2 and the tyrosine kinase domain, alongside with that occurring between the kinase and the SH2 domain, keep c-ABL in a close inactive state [23]. The central part of the protein is characterized by proline-rich (PxxP) stretches acting as docking sites for SH3 containing proteins and a DNA binding domain (DBD). Eventually the carboxy-terminal region contains an actin binding domain (ABD) which allows the interaction either with the monomeric- (G) and with the filamentous- (F) actin. Within cells, the ABL is distributed either in the nucleus and, to a lesser extent, in the cytoplasm where it plays distinct roles. The shuttling between the two compartments it steered by its nuclear-localization and nuclearexport signals and it is depending on different extracellular cues (*e.g.* cell to substratum adherence) [24]. While cytoplasmic c-ABL regulates several actin-dependent cellular processes, for example by positively controlling the filopodia exploration and the membrane ruffling [25], the nuclear c-ABL is a pivotal proapoptotic play-actor, playing a role in the cellular response to genotoxic stress (*e.g.* ionizing radiation) [26]. Alike c-ABL, BCR is a multidomains protein with a peculiarity consisting of a Dbl homology (DH) and a Rho-GAP domain that are localized in the central region and at the C-terminus of the protein, respectively. These domains act as Guanine Exchange Factor (GEF) and GTPase Activating regulatory elements (GAP) for some members of the Rho superfamily, including Cdc42, Rac1, Rac2 and RhoA. Additionally, BCR protein harbors other structural regions, including two lipid binding domains namely Pleckstrin Homology (PH) and Calcium-dependent lipidbinding domain (C2), which is localized in the central part of the protein, and an N-terminal 63 aminoacids long coiled-coil oligomerization peptide that is followed by a Serine/Threonine kinase domain. BCR expression is rather ubiquitous and enriched in brain. Differently from c-ABL, the subcellular localization of BCR is

**234**

*Structural features of the different Bcr-Abl protein variants. Linear depiction of the functional motif composition of the different BCR-ABL proteins: p190, p210 and p230. CC: Coiled-coil; S/T kinase: Serine/ threonine kinase; DH: Dbl-homology; PH: Pleckstrin homology; C2: Ca2+-dependent membrane-targeting module; Rac-GAP: Rac GTPase; SH3: Src Homology3; SH2: Src Homology2; Tyr kinase: Tyrosine kinase: DBD: DNA binding domain; ABD: Actin binding domain; PXXP: Proline rich region, where X indicates any aminoacid.*

features of c-ABL, including SH3, SH2, tyrosine kinase, proline rich regions, DNAbinding and Actin Binding Domains. By contrast, the distinguishing part is represented by the BCR peptide. In the shortest BCR-ABL variant (p190) or alternatively named p185, the BCR portion encodes for a peptide of approximately 490 aminoacids encoded by the first BCR exon, encompassing the very N-terminal coiled-coil oligomerization domain and the serine/threonine kinase domain, fused to the ABL. Conversely, in the longest BCR-ABL variant, p230, the BCR portion harbors all BCR structural domains with the exception of the GAP that is truncated. Eventually, the most common BCR-ABL variant, p210, encodes for a chimeric protein in which the BCR portion comprises the coiled-coil, Ser/Thr, Rho-GEF and PH domains. Crucial for the constitutive activation of the c-ABL tyrosine kinase is the BCR oligomerization domain that promotes either the dimerization or tetramerization of the protein [28]. In this way the BCR-ABL proteins can cross-phosphorylate each other on tyrosine residues in their kinase-activation loops. BCR-ABL phosphorylated tyrosine residues usurp the physiological functions of the normal ABL and can act as docking sites for SH2-domain containing proteins that contribute in activating downstream signaling pathways. On the whole this leads to clear readouts comprising deregulated cellular proliferation, decreased adherence of leukemia cells to the bone marrow stroma and reduced apoptotic response to mutagenic stimuli. Alike the BCR, but differently from the c-ABL protein, strikingly all the BCR-ABL chimeric proteins display a cytoplasmic localization, though all retain both the nuclear-localization and nuclear-export peptide sequences. The main reason for its cytoplasmic localization is its constitutively activated tyrosine kinase activity that thus allows to the chimeric tyrosine kinase to interact and cross talk with a number of proteins, thus exerting its leukemogenic effect. Interestingly, upon BCR-ABL pharmacological inactivation (*i.e.* Imatinib) and concurrent blocking of its nuclear export (*i.e.* leptomycin B) the protein re-localizes within the nuclear compartment and it is trapped there. Astonishingly, upon Imatinib removal and the tyrosine kinase activity of the nuclear BCR-ABL is reactivated it is converted from an antiapoptotic to proapoptotic protein thus inducing cell death [29].
