**2. Role of tyrosine kinase inhibitors in chronic myeloid leukemia treatment**

The principal function of tyrosine kinases (TKs) involves the regulation of multicellular aspects of the organism. By transferring a γ-phosphate group from adenosine triphosphate (ATP) to the hydroxyl group of tyrosine residues on signal transduction molecules, cell-to-cell signals, including growth, differentiation, adhesion, motility, and death, are transmitted [7]. Around 90 TK genes have been identified in the human genome [8]. Based on kinase domain structure, 58 are of transmembrane receptor type and can be grouped into 20 subfamilies and 32 are of cytoplasmic non-receptor type which falls into 10 subfamilies [8, 9]. TK receptors play a role in either transmembranous or intracellular signal transduction as they act as relay points controlling intracellular signaling pathways. Non-receptor TKs exhibit no transmembrane protein domain and are located in the cytoplasm. Generally, they are involved in signaling downstream of the receptor TKs.

In humans, TKs have been demonstrated to play significant roles in the development of many malignant diseases like chronic myeloid leukemia (CML) [8]. CML results from a reciprocal chromosomal translocation involving the c-abl proto-oncogene 1 (*ABL1*) on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22, thus forming the *BCR-ABL1*

oncogene [10, 11]. This t(9; 22) translocation or Philadelphia chromosome (Ph+) is a characteristic cytogenetic abnormality seen in 95% of patients with CML and in 15–30% of adult patients with acute lymphoblastic leukemia (ALL) [12, 13]. The *BCR-ABL1* oncogene codes for two forms of fusion transcripts: p190BCR-ABL1 and p210BCR-ABL1, which are constitutively highly activated and subsequently dysregulate intracellular signaling by enhancing proliferative capability and resistance to apoptosis of hematopoietic stem or progenitor cells, leading to a massive increase in myeloid cell numbers.

children—it was learned that chronic exposure to TKIs impaired modeling of the osseous skeleton as an off-target effect [3]. This skeletal side effect resulted in impaired longitudinal growth in not outgrown minors [4, 5]. With regard to a potential lifelong necessity of TKI intake, children with CML differ from a typical patient with CML who is about 60 years old [6]. Thus, the rational of the research of TKIs' off-target effects is to generate a clear picture of

On this background, the essential objective of this chapter is the description of a juvenile (still growing) rat model that allows a chronic administration of TKIs via the drinking water in order (i) to mimic osseous changes observed in humans, (ii) to further characterize and investigate the causative pathophysiologically mechanisms resulting in impaired bone growth, (iii) to test approaches in growing animals for ameliorating the off-target effect resulting in growth impairment, and (iv) to check further organs beside bone for long-term TKI toxicity. In this chapter, sections describe i) the highly effective role that TKIs play in standardized attempts to operationally cure CML in adults as well as in children, ii) elucidate the role of the established juvenile male Wistar rat model to investigate with ease the skeletal changes at all developmental stages, and iii) focus on the administration of TKI via the drinking water over many weeks as an adequate and convenient way resulting in the achievement of therapeutic drug blood levels. TKI-induced changes in long bones, as well as vertebrae, can be investigated with dedicated small imaging devices while blood levels of bone turnover markers, growth hormone, and vitamin D metabolites can be followed at different stages of development. The results of these investigations as well as the derived hypothesis on the pathophysiological cascade, specifically how TKIs impair longitudinal bone growth, are in excellent agreement with clinical observations. In addition, the juvenile animal model is of value to monitor other long-term TKI side effects on the heart and fertility to generate an overall picture on all possible side effects.

**2. Role of tyrosine kinase inhibitors in chronic myeloid leukemia treatment**

The principal function of tyrosine kinases (TKs) involves the regulation of multicellular aspects of the organism. By transferring a γ-phosphate group from adenosine triphosphate (ATP) to the hydroxyl group of tyrosine residues on signal transduction molecules, cell-to-cell signals, including growth, differentiation, adhesion, motility, and death, are transmitted [7]. Around 90 TK genes have been identified in the human genome [8]. Based on kinase domain structure, 58 are of transmembrane receptor type and can be grouped into 20 subfamilies and 32 are of cytoplasmic non-receptor type which falls into 10 subfamilies [8, 9]. TK receptors play a role in either transmembranous or intracellular signal transduction as they act as relay points controlling intracellular signaling pathways. Non-receptor TKs exhibit no transmembrane protein domain and are located in the cytoplasm. Generally, they are involved in

In humans, TKs have been demonstrated to play significant roles in the development of many malignant diseases like chronic myeloid leukemia (CML) [8]. CML results from a reciprocal chromosomal translocation involving the c-abl proto-oncogene 1 (*ABL1*) on chromosome 9 and the breakpoint cluster region (BCR) on chromosome 22, thus forming the *BCR-ABL1*

early and late sequelae of long-term drug intake.

312 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

signaling downstream of the receptor TKs.

About 1–1.5/100,000 residents are diagnosed with CML every year with an age peak between 50 and 60 years [14], representing around 20% of all cases of leukemia in adulthood [15]. Concerning pediatric patients, the frequency of diagnosis is about 0.05–0.40/100,000 residents per year within the age of 0–18 years [16]. Thus, CML represents one of the rarest leukemic disorders in childhood and adolescent age, accounting for only 2–3% of all children suffering from leukemia [16]. In terms of morphological characteristics, childhood CML is not different from adult CML. However, it is a matter of an ongoing debate whether and to what extend molecular differences exist between CML diagnosed at childhood or older age [6]. For example, pediatric CML shows a breakpoint distribution in the *BCR* gene more similar to adult Ph+ ALL [17].

Still, as the *BCR-ABL1* oncogene is the single molecular aberration causing the development of CML, specific TKIs like imatinib (Gleevec®, Novartis) have been developed to inhibit the BCR-ABL1 TK [19]. By achieving hematological and cytogenetic response in over 90% of the patients after a few months of imatinib treatment, imatinib has been very effective in inhibiting progression of CML (**Figure 1**) [1, 20–23].

**Figure 1.** Survival probabilities by year of diagnosis (1980–2013) of pediatric patients with CML in Germany [18].

However, some patients develop resistance to imatinib resulting in loss of treatment response or even leukemic relapse. Among other underlying mechanisms, BCR-ABL1 kinase domain mutations can cause varying degrees of drug insensitivity [24]. In order to counter these mechanisms, next generation TKIs have been developed like dasatinib (SPRYCEL®, Bristol-Myers-Squibb), nilotinib (Tasigna®, Novartis), bosutinib (BOSULIF®, Pfizer), and ponatinib (ICLUSIG®, Ariad Pharmaceuticals) with different affinities to the ATP-binding pocket of the BCR-ABL1 TK [25].
