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

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 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

*Advances in Precision Medicine Oncology*

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

*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* 

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

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

**238**

induced again [56].

**Figure 2.**

*microenvironment.*

protein encoding genes [59].

special characteristic that is missing in nearly all the other forms of malignancy. Indeed, conversely from other cancers, in which each genotype encodes diverse phenotypic traits, CML displays an unambiguous genotype–phenotype relationship. However, although most of patients responded excellently to Imatinib therapy a minority relapsed. Especially those patients with advanced CML phases initially respond to Imatinib but then progressed to accelerated or blast crisis. The reason for the relapse is straightforward: while in the patients that respond to Imatinib the BCR-ABL tyrosine kinase activity is abrogated, in those that relapse the tyrosine kinase is reactivated due to mechanisms that either prevent Imatinib to reach the target or render the target insensitive to Imatinib. A combination of approaches, including functional studies that have been then validated by the crystallization of the ABL tyrosine kinase domain with Imatinib coupled with the sequencing of the ABL tyrosine kinase domain, allowed to identify and determine critical contact points between the protein and the inhibitor [68, 69]. Indeed, most of the patients who developed Imatinib insensitivity harbor ABL tyrosine kinase point mutation, especially in the P-loop decreasing its flexibility and therefore its capability to bind to Imatinib. The resistance to Imatinib has led to the development of second generation of tyrosine kinase inhibitors (Nilotinib™, Dasatinib™ and Bosutinib™) and the boost of pharmacogenomics [70]. Imatinib is effective also in the treatment of various malignancies, other than CML. For example, it has shown significant activity in patients with Acute Lymphoblastic Leukemia Ph + (ALL Ph+) [71], in a significant proportion of people with Gastrointestinal Tumor (GIST) that harbor c-KIT mutations [72] and those disorders characterized by translocations involving the PDGFRB gene, including myeloproliferative and myelodysplastic syndromes [73, 74]. The demonstration that small molecule inhibitors could effectively treat chronic myeloid leukemia opened the door to the development of new tyrosine kinase inhibitors and to the blooming era of targeted cancer therapies (**Figure 3**).

Though cancer is the predominant indication for tyrosine kinase inhibitors (TKIs), currently the disease targets are extensively growing. For example, Tofacitinib™ is a Jak3 inhibitor that is currently approved for the treatment of rheumatoid arthritis [75, 76] and Nintedanib™ is a FGFR/multikinase inhibitor that is approved for the treatment of pulmonary fibrosis [77]. Furthermore, Pegaptanib, Ranibizumab and Aflibercept that act by inhibiting the VEGF receptor tyrosine kinase activity are currently used for the treatment of the age-related

#### **Figure 3.**

*The FDA approval timeline of tyrosine kinase inhibitors (TKIs): Upon the approval of the Imatinib for the treatment of CML other TKIs have been developed and nowadays small molecules TKIs are dozens. Though, originally they have been designed for neoplasms in the last decade we have also witnessed to an amazingly growth of the diseases, other than cancers, that significantly benefit from TKIs treatment.*

**241**

**Author details**

\*, M. Shahzad Ali<sup>2</sup>

provided the original work is properly cited.

1 Department of Oncology, University of Turin, Italy

\*Address all correspondence to: enrico.bracco@unito.it

, Stefano Magnati2

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Department of Clinical and Biological Sciences, University of Turin, Italy

and Giuseppe Saglio2

Enrico Bracco1

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

number of protein kinases, thus leading to copious side effects.

The authors declare no conflict of interest.

macular degeneration, which is a common cause of visual impairment and blind-

Advances in our understanding in tumor biology have encouraged not only the reassessment of the tumors classification by the site of origin in favor of molecular alterations but also in terms of oncogenic drivers (e.g. tyrosine kinases) amenable for treatment. Since Imatinib has been approved by FDA in 2001 as small molecule competing with ATP, dozens of orally effective small molecule protein kinase inhibitors have been subsequently approved. This is also due to the significantly shortening of the timelines of drug development, as it happened in the case of a record time for the Crizotinib™. The approval of Imatinib for the successful treatment of leukemia (CML) definitively chased away the notion targeting the ATPbinding sites of protein kinases was not selective or efficacious because of the large

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

ness in elderly adult [78–80].

**8. Conclusions**

**Conflict of interest**

macular degeneration, which is a common cause of visual impairment and blindness in elderly adult [78–80].
