Skeletal effects of tyrosine kinase inhibitors

Chronic myeloid leukaemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by a reciprocal translocation between chromosomes 9 and 22 resulting in formation of the Philadelphia chromosome. This chromosomal translocation gives rise to the chimeric BCR-ABL gene. The resulting BCR-ABL fusion protein is a constitutively active, oncogenic tyrosine kinase (TK) that causes cell transformation and CML. Ongoing from the year 2000 targeted therapy with imatinib has been replacing former therapies as frontline treatment for CML, not only in adults but also in children. Imatinib is a TK inhibitor (TK-I) acting by binding to the activation loop of ABL kinase and its derivative BCR-ABL, which traps the kinase in an inactive conformation. In doing so, imatinib inhibits activity of the kinase and in patients with CML leads to an improvement of the median survival from formerly 5 years up to estimated >15 years. However, resistance to imatinib has become of increasing importance as relapse during imatinib treatment is most often caused by mutations in the kinase domain of BCR-ABL thus interfering with imatinib binding. Therefore "second generation" TK-I such as dasatinib and nilotinib were developed, being more active against BCR-ABL and against most of the imatinib resistant subclones. However, all these TK-I are multitarget TK-I which are not highly selective and also bind to other receptor TKs such as cKit, PDGF-R and c-FMS. For example, c-FMS represents the receptor for M-CSF, which after stimulation induces differentiation of blood monocytes into bone resorbing osteoclasts. Bone forming osteoblasts derive from mesenchymal stem cells which undergo differentiation under the control of signalling pathways including PDGF-R and c-ABL. Thus, the inhibitory action of TK-I on TK active in downstream signals cascades may influence bone metabolism by inhibiting proliferation and differentiation of osteoblasts and osteoclast as well as their precursor cells. As a net result the balance between bone resorption and bone formation is changed. In line with these nonselective inhibition of non-BCR-ABL TKs clinical observations on adult patients with CML in imatinib describe hypophosphatemia and changes in bone metabolism by bone turnover serum markers, an increase in trabecular bone volume of iliac crest biopsies in one third to one half of the patients under investigation, as well as an increased bone mineral density as assessed by pQCT. In adult rodents imatinib decreases in vivo the number of bone lacunae as a result of reduced osteoclast activity. CML constitutes approximately 2-3% of all childhood leukemias and only a few studies have specifically addressed CML in childhood. Since licensing of imatinib in 2003 for children also pediatric patients with CML are treated increasingly with this drug responding with rates and side effects similar to adults. Probably due to the small number of cases so far -in contrast to adults-no data from larger series of children with CML on the influence of imatinib on the skeleton have been reported. However, in three single case reports massive longitudinal growth retardation in prepubertal patients with CML under long term imatinib treatment has recently been documented (Mariani S et al. Lancet 2008; 372:111; Kimoto T et al. Int J Hematol; 2009, 89:251; Schmid et al Haematologica 2009;94:1177). Therefore any impact of imatinib on the bone in this not yet outgrown cohort might be of special concern. However, information on the influence of a chronic TK-I intake on the still growing skeletal system from formal standardized trials is presently not available -neither clinically in children and adolescents nor in juvenile animal models. On the one hand the study presented here aims to investigate the influence of three clinical used TK-I imatinb, dasatinib and nilotinib on the growing skeletal system of juvenile rats. Therefore TK-I will be chronically exposed via subcutaneously implanted Azlet Micro-osmotic pumps. The effects on long bones and vertebra will be evaluated by morphological, biochemical, and mechanical assays. In addition, to compare results of the animal model and to draw conclusions about the impact of TK-I on the growing skeleton, the study will examine specific bone metabolic parameters in serum and urine samples, bone age and bone density of pediatric patients with CML while under up-front therapy with TK-I. These patients participate in the study CML-paed II which recruits ~20 patients per annum. A positive statement of the ethics committee of Dresden is available from January, 24 2007 (Nr.: EK 282122006).

In this project, we have characterized further the skeletal side effects of drugs that are commonly used in the therapy of leukemia. The therapy with tyrosine kinase inhibitors (TKIs) has profoundly improved the prognosis of chronic myeloic leukemia (CML). CML is characterized by a genetic translocation resulting in a fusion gene which encodes for the constitutively active BCR-ABL1 tyrosine kinase. Tyrosine kinases have a key role in the regulation of cell proliferation. The therapeutic efficacy of TKIs in CML is based on the inhibition of the BCR- ABL1 tyrosine kinase. Therefore, the therapy needs to be given lifelong. The first approved TKI for therapy in humans was imatinib. In the meantime, other TKIs such as dasatinib and bosutinib have been developed. Unfortunately, besides BCR-ALB1, TKIs also inhibit other physiologically occurring tyrosine kinases, which, amongst other things, participate in bone metabolism. Therefore, TKI therapy is not without side effects. Children with CML suffer from linear growth failure under imatinib treatment by an unknown mechanism. In the current project, we have characterized further the effects of TKIs on bone metabolism in a rat model. To this end, we treated juvenile, fast-growing male rats over 10 weeks with the TKIs imatinib, dasatinib, and bosutinib, starting at 4 weeks of age. The rats were analyzed at the ages of 6, 8, and 14 weeks. We found that continuous exposure to imatinib and dasatinib reduced fem- oral and tibial length, whereas bosutinib did not impair longitudinal bone growth. Intermittent therapy reduced the bony effects of TKI. Because imatinib had the strongest effects on bone, we focussed on imatinib for mechanistic studies. In contrast to our expectations, these stud- ies showed that imatinib did not cause major changes in cancellous bone mass or turnover as measured by histomorphometry in histological sections. However, imatinib induced a dose dependent suppression of longitudinal bone elongation and morphological changes in the growth plates of rapidly growing rats. Interestingly, intermittent therapy with high dose imatinib did not impair longitudinal bone elongation. These data suggest that the linear growth failure under imatinib therapy is mainly caused by a direct drug effect on the growth plates, and not by a body-wide change in bone metabolism. Furthermore, our results suggest that intermittent therapy with imatinib may profoundly reduce the risk of linear growth failure in children with CML.