In Vitro Toxicity of Fludarabine on Normal Bone Marrow (BM) Progenitors Suggests Its Involvement in the Impairment of Hematopoietic Progenitor (HP) Mobilization.
Authors: Richard Céline , Maguer-Satta Véronique , Berger Juliette , Jeanpierre Sandrine , Nicolini Franck E. , Tournilhac Olivier , Michallet Mauricette , Berger Marc G. 
Published: October 2019

Abstract
Fludarabine phosphate (FDR) is considered the most effective drug for treating aggressive B-cell Chronic Lymphocytic Leukemia (B-CLL). Nevertheless, several groups have reported negative effects on BM HP mobilization by G-CSF alone after several months and sometimes correlated with the blood platelet count. As it was previously reported that in vivo FDR-induced cytopenia suggesting toxicity towards HP, we postulate that FDR could impair two major cell components of the bone marrow niche - mesenchymal (MP) and hematopoietic cells - and durably alter the HP egress process. We assessed the effects of increasing doses of FDR (for 5 days) on normal BM MP and HP biological properties, on HP adherence to fibronectin (Fn) or stromal cells and on SDF-1-induced in vitro migration. The expression of molecules involved in HP egress, i.e. CXCR4, CD49d and CD106, was evaluated by flow cytometry. As we demonstrated previously that all MP express CD73, an ecto-nucleotidase probably involved in FDR metabolism, we then tested the effect of a specific CD73 inhibitor (α, b methylene adenosine 5′-diphosphate (MADP)) on MP response to FDR treatment. In two independent series, we found a dose-dependent toxic effect of FDR on BM mononuclear cells, particularly on clonogenic mesenchymal progenitors (MP) (n=8) and hematopoietic progenitors (HP) (n=9). The most sensitive progenitors were MP, BFU-E and CFU-Mk (from 1mM dose) but other progenitors (CFU-GM, CFU-Mix), including the most primitive (LTC-IC) (n=3), were also dose-dependently sensitive. We found that toxicity of FDR on MP was CD73-independent since no improvement in cell survival was observed in presence of MADP (n=4). Interestingly, after expanding the surviving cells, we observed that FDR-induced impairment of the proliferative capacity of input MP was transmitted to cell progeny during the following passages. This means that progeny-derived cells, that have not been directly in contact with FDR, are still affected by the initial dose of FDR in a dose-dependent fashion. In the hematopoietic compartment, FDR had no effect on mononuclear cell adhesion, but there was an increase in the adhesion of HP colony-forming cells (CFC) which correlated with an inhibition of SDF-1 induced migration. However, FDR did not modify the expression of CXCR4, CD49d or CD106 on mesenchymal (CD45CD14) − /CD73+ cells or hematopoietic CD34+ cells. In conclusion, FDR appeared toxic towards clonogenic MP and HP, and profoundly impaired cell metabolism, since the effect persisted in cell progeny. The high sensitivity of the mesenchymal component suggests a possible impairment of BM stem cell niches. Although there was no modification of expression of molecules involved in egress, increased CFC adhesion and inhibition of HP migration suggest a FDR-induced retention of HSC in bone marrow. We are currently evaluating these parameters in MP and HP cells isolated from the bone marrow of CLL patients.