**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 factor stimulation.

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 cellular matrix (**Figure 2**).

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

*Advances in Precision Medicine Oncology*

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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#### **Figure 2.**

*BCR-ABL exerts its leukemogenic effects by impacting diverse cellular processes: the constitutively active BCR-ABL tyrosine kinase triggers a numbers of signaling pathways, including the Ras/Raf/MEK/ERK and PI3K/mTORC/Akt pathways. On the whole their enhanced activation leads to increased cell-survival and –proliferation, and impaired apoptosis rate. Meanwhile, the oncogenic tyrosine kinase impacts also the cellular autophagy rate and eventually the interaction of the BCR-ABL positive leukemic cells with the stromal microenvironment.*

tyrosine kinase activity of BCR-ABL impacts also on the cell-to-substratum adhesion. Indeed, the BCR-ABL-transformed cells display an impaired adhesion to the extracellular matrix. Mostly this behavior is due to the CrkL protein that is one, among many, substrate on the chimeric protein BCR-ABL [42]. Interestingly, CrkL is constitutively binds to BCR-ABL through its first SH3 domain and, at least *in-vitro*, CrkL supports and even potentiates the c-ABL tyrosine kinase activity. CrkL plays a pivotal role in adhesion and cell motility through its association with paxillin, Crk-associated substrate (Cas), Focal Adhesion Kinase (FAK) and the Cbl proto-oncogene. Furthermore, BCR-ABL itself can directly affects the actin cytoskeleton via its actin binding domain localized at its very carboxy-terminal tail and by regulating crucial proteins, such as Rho, Rac and Cdc42 responsible for the cytoskeletal actin dynamics [50–52]. The gene expression of several cell adhesion molecules encoding genes, either mediating the cell-to-substratum and cell-to-cell adhesion including the integrin subunit α-6 and the L- and P-selectins, is under the control of BCR-ABL [53, 54]. Eventually, BCR-ABL suppresses autophagy, an intracellular degradative process allowing cells to adapt to developmental changes and/or unfavorable environmental conditions. Remarkably, autophagy has been shown to provide a survival mechanism to cancer cells [55]. The BCR-ABL-mediated suppression of autophagy occurs via the PI3K/mTORC/Akt signaling pathway since by pharmacologically inhibiting the PI3K in BCR-ABL expressing cells the autophagy is induced again [56].

Alike CrkL some downstream BCR-ABL downstream effectors might play dual role, such is the case of Stat5 that is directly tyrosine phosphorylated by BCR-ABL in a JAK independent way [57, 58]. The Stat5 transcription factor mediates the transcription of several pro-survival and pro-proliferative, as well as anti-pro-apoptotic protein encoding genes [59].

Interestingly, though all BCR-ABL variant proteins are collectively characterized by constitutive and enhanced tyrosine kinase activity they still differ in their binding partners, substrates and as a consequence in their elicited signals. For example, while both p190 and p210 can activate the Ras/Raf/MEK/ERK through the Grb2/SOS complex that binds to the phosphorylated tyrosine residue at position 177 (Tyr-177), the activation of Stat5 is exclusively triggered by the p210. Conversely, the p190, when compared to the p210, shows higher affinity towards the tetrameric

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

Adaptor Protein Complex 2 (AP2), the adaptor protein DOK1 and the tyrosine kinase Lyn [60]. Overall, the signals triggered by the constitutively active BCR-ABL tyrosine kinase are promiscuous affecting several aspects of the components of the

**7. BCR-ABL inhibitors: paving the way for novel tyrosine kinase** 

animals, in which the genes encoding for tyrosine kinases were inactivated, displayed embryonic lethal phenotypes. Altogether these observations led to the acceptance that tyrosine kinase inhibitors would have been enormously toxic, thus inadequate either for scientific and clinical use. Last but not least, it was assumed that the selective targeting of a single defect would not be sufficient to treat a highly heterogeneous disease such cancers. However, by the end of the 1980s and the beginning of 1990s the first selective tyrosine kinase inhibitors (TKIs) were developed, tyrphostins also known as benzene malononitrile derivatives. Outstandingly, these compounds were found to effectively inhibit EGFR [61]. Afterwards, with crucial reagents in hands, including phospho-tyrosine specific antibody coupled to time-consuming approach such as high-throughput screens of chemical libraries, a team of scientists at Giba-Geigy (now Novartis) seeking for compounds with kinase inhibitory activity identified a promising class of compounds: the 2-phenylaminopirimidine series. Surprisingly, among these molecules one displayed very high selectivity towards the receptor for the platelet derived growth factor (PDGFR), ABL and the stem cell factor receptor (c-kit) [62, 63]. In the first half of 1990s a single molecule the Signal Transduction Inhibitor 571 (STI571) (Gleevec, Glivec, Imatinib™) was shown to be the most specific at selectively suppress the growth of BCR-ABL expressing cells either from CML patients or cell lines [64]. Afterwards, following these *in-vitro* encouraging results pre-clinical data were produced with the help of BCR-ABL transgenic animal models [65, 66]. Consistent with the "*in-vitro*" evidence the animal studies showed that Imatinib treatment led to a dose dependent selective inhibition of BCR-ABL-expressing cells without significant effects against v-SRC-expressing tumors. On the whole, in 1998 these encouraging and promising data prompted a handful of scientist led by B. Druker to set-up the first clinical trial using Imatinib in CML patients. However, before clinical trials could start scientists had to overcome some difficulties concerning the toxicity of the molecule, whether targeting a single kinase would have been an effective and successful strategy and most important whether the pharmaceutical company would realize a return on its investment due to the fact that CML is a pretty rare disease and thus representing a small market. At the end of the 1990s a phase I dose escalation study using Imatinib in CML patients' refractory to IFNαbased therapy started. Surprisingly, within one year the vast majority chronic phase patients who had failed IFN-α therapy and treated with Imatinib 300 mg once a day achieved a complete hematological response. These promising data paved the way for a phase II study and eventually in 2001, three years later after the phase I, Imatinib received the final approval from the Food and Drug Administration (FDA) [67]. The dramatic success in the treatment of CML by an inhibitor of the BCR-ABL kinase is due to a mechanism involving a single biochemical defect a

Amazingly, in the 1980s and 1990s both the scientific community and pharmaceutical industry were rather skeptic about the issue of pharmaceutically inhibiting protein kinases. Much of their skepticism was lying in the prevailing perception that ATP-binding competitive inhibitors would have had a rather limited target specificity to be translated into useful clinical drug. Moreover, some of the early transgenic

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

cellular machinery.

**inhibitors**

Adaptor Protein Complex 2 (AP2), the adaptor protein DOK1 and the tyrosine kinase Lyn [60]. Overall, the signals triggered by the constitutively active BCR-ABL tyrosine kinase are promiscuous affecting several aspects of the components of the cellular machinery.
