Dasatinib Exerts Differential Effects on Normal and BCR-ABL Positive Hematopoietic Cells in a Transgenic Mouse Model of Chronic Phase-CML
Authors: Schubert Claudia , Chatain Nicolas , Braunschweig Till , Schemionek Mirle , Feldberg Kristina , Hoffmann Melanie , Brümmendorf Tim H. , Mustjoki Satu , Dufva Olli , Koschmieder Steffen 
Published: October 2019

Abstract
The second generation tyrosine kinase inhibitor dasatinib is a clinically approved drug for chronic myeloid leukemia (CML) as well as Ph+ acute lymphoblastic leukemia. Dasatinib is a dual-specific inhibitor of ABL and SRC family kinases but shows a broad inhibitory effect on various kinases, including Kit, EGFR, FAK and BTK. In addition to its antileukemic effects, dasatinib was shown to impact on the immune system, including alterations in B-cell development, T-cell differentiation, as well as a transient activation of quiescent hematopoietic stem cells (HSCs) in the bone marrow of wild-type mice. In CML patients treated with dasatinib, the development of pleural effusions and large granular lymphocyte (LGL) lymphocytosis during dasatinib treatment was associated with a favorable response. Non-hematologic adverse events included pulmonary artery hypertension and gastrointestinal symptoms.
We investigated the influence of dasatinib (5 or 20 mg/kg p.o.) on the cellular composition of immune cells in our tetracycline-controlled transgenic BCR-ABL mice in comparison to control mice. We analyzed dasatinib-induced effects by flow cytometry in the bone marrow (BM) and spleen under steady-state conditions as well as after bone marrow transplantation. Furthermore, we conducted flow cytometry analysis of stem- and progenitor cells in the BM of BCR-ABL expressing mice treated with dasatinib or vehicle control and performed histophathology to evaluate effects on the BM, spleen, lung and intestine.
Our results demonstrated that dasatinib dose-dependently increased Gr1/CD11b positive myeloid cells in the BM of normal mice, while B220 positive B cells and Ter119 positive erythrocytic cells were reduced. NK1.1 positive NK cells were decreased in BM but increased in the spleen. Interestingly, the lower dose (5 mg/kg) induced an increase of the percentage of CD41 positive megakaryocytic cells in BM and B cells in the spleen, while higher doses (20 mg/kg) decreased both percentages. The BCR-ABL-induced phenotype was strongly reversed by dasatinib in BCR-ABL expressing transgenic animals under steady-state conditions and in a transplantation setting: the oncogene-induced upregulation of granulocytes, megakaryocytes, and the reduction of erythroid cells as well as of B-lymphocytes was antagonized by dasatinib. Concurrently, a strong decrease of Linneg Sca1+ c-kit+ (LSK) HSCs and an increase of MEPs together with a reduction of GMPs were observed. Although the overall lymphocyte population was reduced in the BCR-ABL-expressing mice and replaced by granulocytes, we observed an increase of the proportion of CD3+, CD4+ and CD8+T cells among lymphocytes, and this increase was reverted by dasatinib treatment. Conversely, dasatinib did not significantly alter NK1.1 cells in these mice. Our observations in BCR-ABL-positive animals are in part contrary to the findings in wild-type animals, but this may be explained by the reversion of BCR-ABL effects in addition to the effects of dasatinib on normal hematopoietic cells. In addition to the above-mentioned changes, we observed a reduction of BCR-ABL mediated granulocyte infiltration of the small intestine indicating a beneficial effect in attenuating inflammation of the bowel. There was no significant alteration of the bronchial epithelium or bronchial arteries.
Altogether, dasatinib robustly reduced the CML phenotype in vivo in our transgenic mouse model, and this effect included stem and progenitor populations and was stronger than the effects we have previously described for imatinib in this model. Thus, our transgenic CML mouse model is well suited to examine dasatinib effects on normal and malignant hematopoietic cells in vivo. Interestingly, there was a differential effect on myeloid cells in normal vs. CML mice, suggesting different targets in these cells. (CS and NC contributed equally to this study)

Disclosures
Braunschweig: Bristol-Myers-Squibb: Honoraria; MSD Sharp & Dome: Honoraria. Brümmendorf:Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Koschmieder:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel funding to conferences, Research Funding.