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

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

immortalized fibroblast cell lines, growth-factor-dependent hematopoietic cell lines, primary bone marrow cells and mice. Though all these models represent very important tools that have significantly contributed to elucidate the molecular mechanisms of CML formation and to identify potential therapeutic targets, each of them display pros and cons either in term of their tractability and physiological relevance. Many cancer cell lines, including leukemia, have been excellent models for "*in-vitro*" studies because of their relative ease in obtaining a large number of cells for biochemical analysis, genetic manipulation and biological examinations. However, they display remarkable limitations, including their failure to recapitulate the physiology of the disease. By contrast animal models are excellent in term of physiological relevance, thus allowing to recapitulate the disease and to assess its potential evolution, but rather deficient in tractability. The product of BCR-ABL is a constitutively active tyrosine kinase that is more active than c-ABL, thus the expression of BCR-ABL transforms established mouse fibroblast cell lines, factordependent hematopoietic cell lines and primary bone-marrow cells. Usually, under physiological conditions normal hematopoiesis requires a strict balancing among cellular-proliferation, −growth and –survival, which are all tightly regulated by growth factors and cytokines (e.g. IL-3, IL-7, GM-CSF and erythropoietin) [30], which upon binding to their cognate receptors activate a number of intracellular signaling pathways. By making use of different cell lines it has been determined that the constitutively active BCR-ABL tyrosine kinase abrogates this growth factor dependency [31] by activating essential downstream molecules in a ligand independent manner. Hence, the expression of BCR-ABL, likewise v-ABL, confers immortalizing properties to the cells. In summary, cellular models have been extremely useful to dissect the molecular pathways activated by BCR-ABL and to determine which parts of the protein are required to confer transforming properties. Nonetheless, transgenic murine models offer additional benefits thus allowing to ascertain and further validate which parts of the protein are mandatorily required for the induction of a CML-like disease, to study the role of the environment in leukemogenesis and eventually to identify therapeutic target for pre-clinical investigations. The "*in-vivo*" convincing experimental evidence validating the leukemogenicity of BCR-ABL were provided only around the 1990s by using transgenic murine models [32, 33]. In this respect, the initial development of transgenic and knock-in murine CML models displayed major drawbacks. Indeed, the generation of conventional BCR-ABL transgenic knock-in mice, through the expression of the chimeric gene under the control of the BCR promoter, caused embryonic lethality due to the toxicity of the activated tyrosine kinase during embryonic development. Afterwards, the use of murine stem-cell retroviral vector and mice created through expression of BCR-ABL under the control of a tetracycline-responsive promoter allowed to overcome that problem and revealed that to develop a CML-like disorder it is crucial to express this oncogene in proper tissue/cell type. With the help of these models it was also shown that the expression of the p210 BCR-ABL variant in bone marrow caused a CML-like disease. Remarkably, the progression of the p210 associated disease was consistent with the apparent indolence of the human CML chronic phase. Interestingly, mice models expressing the p190 variant at levels similar to that of the p210, allowed to uncover that they displayed clinically distinct conditions consisting in a de-novo development of acute leukemia with a short period of latency [34]. Furthermore, these studies allowed to functionally dissect the BCR-ABL protein and to determine to what extent the different domains of the BCR-ABL protein are required for the onset of the different kind of leukemia. The tyrosine-kinase activity of BCR-ABL is essential for its oncogenic properties, but not sufficient. Indeed, although the transduction of v-ABL in a helper viruscontaining system causes a murine hematopoietic disease it is distinct from the

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factor stimulation.

cellular matrix (**Figure 2**).

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

CML-like syndrome elicited by BCR-ABL developing only modest splenomegaly and malignant disease of several hematopoietic cell types [35]. In addition to its tyrosine kinase domain the *in vivo* molecular dissection of the protein led to the identification of another domain that is apparently required for the induction of the CML-like disease. This domain turned out to be the SH2. Remarkably, the SH2 domain requirement is peculiar only for myeloid but not for the lymphoid leukemogenesis. Notably, this later issue has been rather debated due to some discrepancies between different studies [36, 37]. Additionally, in mice model for the induction of the chronic leukemia-like disease the Grb2 binding site (Tyr-177) is required [38].

**6. Molecular mechanisms conferring oncogenic properties to BCR-ABL**

Once BCR-ABL has been identified as the molecular pathogenic event in CML and other leukemia related disorders, significant effort has been addressed to unveil the molecular mechanisms of action of the chimeric tyrosine kinase through the identification of signaling pathways that are impacted by BCR-ABL. The most prominent feature of the BCR-ABL fusion protein is its potent and constitutive tyrosine kinase activity. The tyrosine phosphorylation is a vital mechanism of intracellular signal transduction, used by many growth factor receptors. Usually, approximately less than 2% of total cellular tyrosine residues are phosphorylated, and the activity of tyrosine kinases is counterbalanced by the activity of tyrosine phosphatases. In cells that express a constitutively active tyrosine kinase, this finelytuned regulation is subverted, leading to a situation that resembles chronic growth

Actually, BCR-ABL displays a tyrosine kinase activity amazingly higher than that of to the c-ABL counterpart [39] and differences among the different variants have been assessed, being the p190 more potent than that of the p210 and the latter more potent than p230 [40]. Though the BCR-ABL oncoprotein can activate a large number of different signal transduction pathways they appear to target few crucial cellular functions, including increased cellular proliferation, reduced apoptosis and autophagy combined with a deregulated interaction with the bone marrow stromal

Whereas in BCR-ABL transformed cell the PI3K/AKT signaling has been shown

to have a pivotal role in mediating both the activation of cell survival and antiapoptotic signaling, the activation of the Ras/Raf/MEK/ERK cascade has been implicated in the BCR-ABL-dependent uncontrolled cell growth [41]. To the latter purpose the adaptor protein Crk Like (CrkL) has shown to be an important player, being constitutively bound to and a substrate of BCR-ABL [42, 43]. Noteworthy, BCR-ABL itself, through the phosphorylated Tyr-177 can activate the Ras/Raf/ MEK/ERK pathway by interacting with Grb2 which in turn recruits SOS that activates Ras [44, 45]. Eventually, Ras triggers the downstream signaling cascade leading to the activation of ERK1/2 [46]. The BCR-ABL dependent pathways leading to apoptosis resistance involve the aberrant expression of the apoptosis regulators proteins of the Bcl2 family including Mcl1, Bcl2 and BclXL along with the proapoptotic members Bim and Bad [47, 48]. Their regulation is mediated by the BCR-ABL-activated PI3K/AKT pathways [49]. The AKT-dependent phosphorylation of Bad leads to its dissociation from Bcl2 and to its sequestering by the adaptor protein 14-3-3, hence leaving less free Bad available to heterodimerize with the antiapoptotic BclXL proteins. Therefore, more BclXL and Bcl2 remain in the cytoplasm exerting their antiapoptotic role by preserving the mitochondria outer membrane integrity. In addition, it is likely that BCR-ABL also negatively regulates c-ABL, whose function in regulating the apoptotic process is central. The constitutive

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

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

CML-like syndrome elicited by BCR-ABL developing only modest splenomegaly and malignant disease of several hematopoietic cell types [35]. In addition to its tyrosine kinase domain the *in vivo* molecular dissection of the protein led to the identification of another domain that is apparently required for the induction of the CML-like disease. This domain turned out to be the SH2. Remarkably, the SH2 domain requirement is peculiar only for myeloid but not for the lymphoid leukemogenesis. Notably, this later issue has been rather debated due to some discrepancies between different studies [36, 37]. Additionally, in mice model for the induction of the chronic leukemia-like disease the Grb2 binding site (Tyr-177) is required [38].
