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Description
Cancer continues to be a leading cause of death worldwide. This disease also challenges oncology pharmaceutical development programs determined to reduce undesirable side effects associated with many cancer treatments. Bone marrow toxicity is a common dose limiting adverse effect of many cancer therapies, resulting in immunosuppression or anemia that may require expensive growth factor treatment and/or dosing holidays, and may also limit treatment combinations. Many kinase inhibitor drugs used for cancer treatment can cause bone marrow toxicity. The main objective of this study was to establish and validate a new model system to identify key kinases associated with bone marrow toxicity. This was accomplished by using siRNA-mediated gene knockdown in human bone marrow mononuclear cells (MNCs) and CD34+ progenitor cells of tyrosine protein kinases that are known to play a role in hematopoiesis. After kinase gene knockdown, cell proliferation effects were assessed to see if the kinase played a key role in the cell proliferation process. Among the target kinases studied was Janus kinase (Jak) 2 which is a well characterized for its role in hematopoietic signaling and non-redundant function in mediating cytokine receptor signaling. Electroporation was used to introduce siRNA, and results were compared to negative controls (siRNAs with sequences that do not target any gene product) under the same electroporation conditions. At 48 hours post electroporation more than 75% knock down of Jak2 was confirmed at the mRNA level using real-time quantitative PCR in MNCs and CD34+ progenitor cells. Jak2 knockdown resulted in a 50% reduction in cell count in MNCs and a 40% reduction in CD34 + progenitor cells that were induced to granulocyte, erythrocyte, monocyte, and macrophage cell lineages. An even further decrease (60% reduction in cell number) was seen in CD34 + progenitor cells that were induced to undergo differentiation into a specific erythroid cell lineage. Suppression of other tyrosine protein kinases that play a role in hematopoiesis, including c-Kit, Axl, colony stimulating factor 1 receptor (CSF-1R) and Lyn, were also evaluated in this model. C-Kit knockdown lead to a decrease in cell proliferation in MNCs and CD34 + progenitor cells whereas Axl, Lyn, and CSF-1R knockdown did not decrease in cell proliferation. These observations may be explained by the fact that many kinases have redundant roles in the hematopoietic process. Overall, this model system could be used to identify key kinases associated with bone marrow toxicity in the early stages of drug development where novel kinase can be evaluated for potential bone marrow risks.
