Abstract
The aberrant tyrosine phosphorylation, either due to constitutive tyrosine kinases (TKs) or to inactivation of protein tyrosine phosphatases (PTPs), is a widespread feature of many cancerous cells. The BCR-ABL fusion protein, which arises from the Philadelphia chromosome, is a molecular distinct and peculiar trait of some kind of leukemia, namely Chronic Myeloid and Acute Lymphoblastic Leukemia, and displays constitutive tyrosine kinase activity. In the chapter, we will highlight the milestones that had led to the identification of the BCR-ABL fusion gene and its role as the only molecular pathogenic event sufficient to elicit and sustain chronic myeloid leukemia. We will also discuss the effort made to unveil the molecular mechanisms of action of the chimeric tyrosine kinase that eventually lead to aberrant cell proliferation and impaired cell-death. Furthermore, we will also review the lesson learned from the selective inhibition of BCR-ABL which currently represent a breakthrough in the treatment of several tumors characterized by defective tyrosine kinase activity.
Keywords
- chromosomal translocation
- fusion gene
- tyrosine kinase
- leukemia
- tyrosine kinase inhibitors
- targeted therapy
1. Introduction
The up-regulated enzymatic activity of tyrosine kinases (TKs) is one of the most frequent events in human cancers. Basically, it is attributable to three distinct molecular-genetic mechanisms covering either the overexpression, the activating mutations or eventually chromosomal translocations involving tyrosine kinase genes. Therefore, targeting the kinases harboring oncogenic properties has led to prominent changes in cancer clinical management. An outstanding contribute in achieving the goal has been offered by the BCR-ABL oncogene, whose story started more than half a century ago. In the 1960s a couple of scientists working in Philadelphia described a major chromosomal abnormality in patients affected by Chronic Myeloid Leukemia (CML) [1, 2]. The chromosomal aberration consisted of an acrocentric chromosome that was originally thought as the outcome of a chromosomal deletion. At that time, it was the first chromosomal abnormality unambiguously associated to a specific malignancy. With the improvement of the chromosomal banding techniques, it became clear that the chromosome abnormality was a shortened chromosome 22. Among a chorus of skepticism and wonder at the beginning of the seventies that short chromosome, that it is now known as Philadelphia chromosome (Ph), was identified as the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22). However, we had to wait until the eighties to know that the exact molecular consequence of the t(9;22) was a fusion gene encoding for a chimeric protein displaying constitutively tyrosine kinase activity. Altogether these discoveries delivered an outstanding message whereby a disease was tightly linked to a single oncogene, BCR-ABL. Since then, dozens of translocations have been found in other cancers, including acute promyelocytic leukemia.
BCR-ABL is a peculiar protein for several of reasons: 1) it is a chimeric protein that is encoded by a fusion gene deriving from a reciprocal chromosomal translocation; 2) it is a constitutively active tyrosine kinase eliciting oncogenic signals, 3) it has been the first oncogene associated to a disease displaying dual properties either as driver and in sustaining the neoplasm evolution, and 4) it has been the first kinase to be selectively targeted with small molecules, thus paving the way for the development of a number of tyrosine kinase inhibitors (TKIs).
In the present chapter we are going to discuss the milestones of a story, started 60 years ago, which has happily led to the selective pharmacological inhibition of BCR-ABL. Hence, CML, whose diagnosis was before a death sentence, is now successfully cured in the vast majority of the cases.
2. Is there any specific reason underlying the generation of the Ph chromosome?
Basically, every chromosomal translocations require DNA Double-strand breaks (DSBs) in two different locations and that the broken ends of non-homologous chromosomes are fused together. DNA double-strands breaks might be due to different causes (
Aside these notions, currently our knowledge regarding the molecular mechanisms responsible for the reciprocal chromosomal translocation occurring between the chromosome 9 and the 22, t(9;22), generating the Philadelphia chromosome (Ph), remain still rather elusive. Fundamentally, it has been speculated that there are two plausible hypotheses. One view prefers to lean towards an entirely random “breaking and re-ligating process” occurring with relatively similar frequency between any two chromosomes within a cell. Chromosomal translocations that give and adaptive advantage are pretty rare and associated with negative consequences (
3. What is the consequence of the t(9;22)?
The results of the chromosomal translocation occurring between the chromosome 9 and 22, t(9;22), are a longer chromosome 9 (9q+) and a smaller derivative chromosome 22, the Ph [7, 8]. By the eighties of the past century the molecular characterization of the Ph led to the identification of a novel chimeric gene, BCR-ABL, which later on has been found to encode for a chimeric protein with a constitutive tyrosine kinase activity and with potent oncogenic properties [9, 10]. The c-ABL and Breakpoint of Cluster Region (BCR) loci are localized on the long arm of the chromosome 9 and 22, respectively [11]. Depending on the different breakpoints occurring on the two chromosomes resulting in different BCR-ABL variants. Though, all BCR and c-ABL DNA breakpoints fall within intronic regions those occurring in the BCR gene are highly variable and thus responsible for defining the major differences among the different variants. The variation in the BCR part of the fusion transcript contrasts with the constant c-ABL part. Indeed, all the breakpoints so far identified within the c-ABL gene occur in a large (300-kb) region in the 5′ portion of the gene, localized upstream of the exon 2, and generally falling in the intron sequences restricted between the two alternative first exons (1b and 1a). Regardless the structure of the different fusion genes the BCR exons directly fuse to the second c-ABL exon (a2). The most frequent BCR-ABL fusion variant is the p210 in which the BCR exon 13, or 14, is fused downstream of the alternative exons 1 of the c-ABL gene and thus leading to a fusion protein with approximately the first half from BCR and the remaining second half from ABL. Mostly this variant is found in CML patients accounting for approximately 95% of the BCR-ABL fusion gene in all the CML cases. A second frequent variant, p190, is found in approximately 20–30% of adult patients with Acute Lymphoblast Leukemia (ALL) [12] and, very rarely, also in Acute Myeloid Leukemia (AML) [13]. When compared to the p210 variant, in this case the breakpoint within the BCR locus is localized in the 3′ half of the first BCR intron, thus encoding for a shorter BCR portion (approximately 425 aminoacids). The third most common BCR-ABL variant, p230, is the largest and is defined by a breakpoint cluster region encompassed between the exons 19 and 21. Whereas the p190 characterizes a more acute form of leukemia usually of lymphoid origin, the latter variant is peculiar of neutrophilic CML. Besides, there are additional BCR-ABL variants, though they have been observed less frequently. Interestingly, some of them are peculiar because they are the results of alternative splicing leading to truncated chimeric proteins that are all lacking tyrosine kinase activity [14]. Furthermore, in hematopoietic malignancies, the BCR gene has been identified 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, 21, 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
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 nuclear-export signals and it is depending on different extracellular cues (
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 lipid-binding 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 features of c-ABL, including SH3, SH2, tyrosine kinase, proline rich regions, DNA-binding 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 (
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 “
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 finely-tuned 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 anti-apoptotic 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 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
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 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.
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
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 macular degeneration, which is a common cause of visual impairment and blindness in elderly adult [78, 79, 80].
8. Conclusions
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 ATP-binding sites of protein kinases was not selective or efficacious because of the large number of protein kinases, thus leading to copious side effects.
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